METHODS OF ISOLATION OF CELL FREE COMPLEXES AND CIRCULATING CELL-FREE NUCLEIC ACID

BACKGROUND

The majority of nucleic acids (DNA and RNA) in the body are located within cells. However, extracellular nucleic acids can also be found circulating in the bloodstream. Such circulating, cell free nucleic acid may be the result of active, spontaneous release of newly synthesized nucleic acids from the cell or the result of release from necrotic and apoptotic cell death (Stroun M, et al., (2001) Clin Chim Acta 313: 139-142; van der Vaart M, et al., (2008) Ann N Y Acad Sci 1137: 18-26).

Circulating, cell free nucleic acid offers an unprecedented non-invasive approach to a wide range of diagnostics for clinical disorders not only in predictive and proactive medicine but also in personalized medicine. Further, circulating, cell free nucleic acid can offer unique opportunities for early surveillance of disease onset, e.g. in early cancer detection (Gormally E, et al., (2004) Int J Cancer 111: 746-749; Fleischhacker M, et al., (2007) Biochim Biophys Acta 1775: 181-232; Frattini M, et al., (2008) Cancer Lett 263: 170-181; Schwarzenbach H, et al., (2008) Ann N Y Acad Sci 1137: 190-196). The ability to isolate, quantify, and analyze circulating, cell free nucleic acid has led to the identification of disease-specific aberrations such as chromosomal abnormalities, gene mutations, methylation and copy number variations which are indicative of diseased cells (Diehl F, et al., (2005) Proc Natl Acad Sci USA 102: 16368-16373; Allegra C J, et al., (2009) J Clin Oncol 27: 2091-2096; Sunami, E.; et al., (2009) Methods Mol. Biol. 507, 349-356; Lu, Y. et al., (2013) PLoS One 2013, 8, e63056). It is believed that over the next decade circulating, cell free nucleic acid will become a non-invasive, standard-of-care for the determination of molecular markers in cancer management. Of particular interest is the growing belief that the analysis of circulating, cell free nucleic acid may provide a more global picture beyond the abnormalities and heterogeneity presented in the primary tumor tissue.

Circulating, cell free nucleic acid can be used as a disease biomarker in a number of ways. Increases and/or decreases in concentration of circulating, cell free nucleic acid may indicate the presence of a disease or the predisposition to a certain disease. Furthermore, the presence of tumor-specific mutations, gene expression signatures or other genomic signatures (for example, methylation patters) may also indicate the presence of a disease or the predisposition to a disease. In addition, the overall expression profile may also be used. Although a number of methods for extraction of circulating, cell free nucleic acid are known, each of the methods of the prior art suffer from certain deficiencies. Generally, the efficiency and quantification of circulating, cell free nucleic acid by the methods of the prior art is variable due to lack of normalization of the experimental conditions. Further, a significant portion of circulating, cell free nucleic acid obtained by the methods of the prior art is highly fragmented with the average size of fragments ranging 100-500 bp in the case of DNA (Mouliere F, Lu, Y. et al., (2013) PLoS One 2013, 8, e63056 (2011) PLoS ONE 6(9): e23418). Importantly, the methods of the prior art isolate free nucleic acids that are not associated with additional cellular components. Such an approach is less than optimal as the removal of co-associating cellular components represents a loss of valuable information. Such approaches also fail to target nucleic acids that are associated with polypeptide and other components that indicate the isolated nucleic acids are active physiologically and relevant to disease diagnosis and treatment. Importantly, nucleic acids associated with other cellular components are protected from degradation in the circulation, allowing the isolation of longer nucleic acids containing more information.

Accordingly, it is desirable to develop novel technologies that can consistently and selectively enrich, in high yield and purity, protected, higher-molecular-weight, biologically relevant circulating, cell free nucleic acid and associated cellular components. Such circulating cell free nucleic acid and associated cellular components may then be used in a variety of analytic, diagnostic and other approaches to indentify genomic, proteomic and lipidomic characteristics for disease diagnosis, prognosis, treatment and monitoring. The present disclosure provides such methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a simplified flow chart illustrating the steps performed in one embodiment of the methods described.

FIG. 1B shows two exemplary chromatograms on the material isolated from plasma samples obtained from a subject using the methods described. Chromatograms were obtained by monitoring ultraviolet absorbance at 260 nm.

FIG. 2 shows chromatograms (obtained by monitoring ultraviolet absorbance at 260 nm) illustrating the effect of pre-treatment of the plasma samples with DNase, DNase and RNase and proteinase K before density gradient centrifugation by the methods described herein.

FIG. 3 shows agarose gels illustrating the effect of pre-treatment of the plasma samples with DNase, DNase and RNase, mung bean nuclease and proteinase K before density gradient centrifugation by the methods described herein.

FIG. 4 shows agarose gels of circulating cell free DNA isolated using the methods of the present disclosure and two commercially available extraction kits.

FIG. 5 shows representative results of array comparative genome hybridization of select individual chromosomes using circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available Kit Q of Example 4 and reference genomic DNA.

FIG. 6 shows the results of array comparative genome hybridization of each autosome and allosome using circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available Kit Q of Example 4 and reference genomic DNA.

FIG. 7 shows the identification of lipid components associated with the isolated circulating cell free nucleic acid.

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to centrifugation and isolating the cell free complex. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a second aspect, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to density gradient centrifugation and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a third aspect, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a fourth aspect, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to cesium-chloride (CsCl) density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a fifth aspect, the present disclosure provides a method for the detection of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and associated cellular components and detecting the cell-free nucleic acid, the associated cellular component or a combination of the foregoing. Such cellular components include, but are not limited to, polypeptides and lipids. Such nucleic acid may be DNA and/or RNA.

In a sixth aspect, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and analyzing the cell-free nucleic acid, associated cellular components or a combination of the foregoing to determine a characteristic of the nucleic acid, associated cellular components or a combination of the foregoing. Such cellular components include, but are not limited to, polypeptides and lipids. Such nucleic acid may be DNA and/or RNA.

In a seventh aspect, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and analyzing the cell-fee nucleic acid to determine a nucleic acid characteristic. The method may further comprise analyzing a cellular component. Such additional cellular components include, but are not limited to, polypeptides and lipids. Such nucleic acid may be DNA and/or RNA.

In an eighth aspect, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and analyzing the associated polypeptide component to determine a polypeptide characteristic. The method may further comprise analyzing a nucleic acid component and/or an additional cellular component. Such additional cellular components include, but are not limited to, lipids. Such nucleic acid may be DNA and/or RNA.

In a ninth aspect, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and analyzing the associated lipid component to determine a lipid characteristic. The method may further comprise analyzing a nucleic acid component and/or an additional cellular component. Such cellular additional components include, but are not limited to, polypeptides. Such nucleic acid may be DNA and/or RNA.

In tenth aspect, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and analyzing the cell-free nucleic acid to determine the presence of a mutation, the presence of a polymorphism, the methylation state, the concentration of a nucleic acid, the level of expression of a nucleic acid or a combination of the foregoing. The method may further comprise analyzing a cellular component. Such cellular components include, but are not limited to, polypeptides and lipids. Such nucleic acid may be DNA and/or RNA.

In an eleventh aspect, the present disclosure provides a method for determining a biologically relevant profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and identifying a plurality of nucleic acids or associated cellular components to produce the profile. Such method may further comprise comparing the subject profile to a corresponding profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding profile indicative of a disease state. The profile may be a nucleic acid profile, a polypeptide profile, a lipid profile or a combination of the foregoing. Such nucleic acid may be DNA and/or RNA.

In a twelfth aspect, the present disclosure provides a method for determining a biologically relevant nucleic acid profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and identifying a plurality of cell-free nucleic acids to produce the nucleic acid profile. Such method may further comprise comparing the subject profile to a corresponding nucleic acid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding nucleic acid profile indicative of a disease state.

In a thirteenth aspect, the present disclosure provides a method for determining a biologically relevant polypeptide profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and identifying a plurality of polypeptides in the associated cellular components to produce a polypeptide profile. Such method may further comprise comparing the subject profile to a corresponding polypeptide profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding polypeptide profile indicative of a disease state.

In a fourteenth aspect, the present disclosure provides a method for determining a biologically relevant lipid profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and identifying a plurality of lipids in the isolated associated cellular components to produce a lipid profile. Such method may further comprise comparing the subject profile to a corresponding lipid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding lipid profile indicative of a disease state.

In a fifteenth aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and determining the presence of a characteristic associated with the disease in the components of the complex. Such characteristic may be the presence of nucleic acid characteristic, a proteomic characteristic or a lipid characteristic.

In a sixteenth aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and determining the presence of a nucleic acid characteristic in the cell-free nucleic acid, wherein the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing.

In a seventeenth aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and determining the presence of a polypeptide and/or lipid characteristic in the cellular component, wherein the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such polypeptide characteristic includes, but is not limited to, a mutation, the presence of post-translational modifications, the presence of insertions or deletions, the concentration, level of expression of a nucleic acid, the profile of the polypeptides or a combination of the foregoing. Such lipid characteristic includes, but is not limited to, the presence of altered forms, the presence of modifications, the concentration, the expression level, the profile of lipids or a combination of the foregoing.

In an eighteenth aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising generating a nucleic acid, polypeptide and/or lipid profile from a subject as set forth in the twelfth to fourteenth aspects and comparing the generated profile to a corresponding disease fingerprint and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the disease fingerprint.

In an nineteenth aspect, the present disclosure provides a method for the isolation of circulating, cell-free nucleic acid, the method comprising subjecting a sample containing the circulating, cell-free nucleic acid to centrifugation and isolating the circulating, cell-free nucleic acid. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twentieth aspect, the present disclosure provides a method for the isolation of circulating, cell-free nucleic acid, the method comprising subjecting a sample containing the to density gradient centrifugation and isolating the circulating, cell-free nucleic acid from the gradient. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-first aspect, the present disclosure provides a method for the isolation of circulating, cell-free nucleic acid, the method comprising subjecting a sample containing the circulating, cell free nucleic acid to density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the circulating, cell free nucleic acid from the gradient. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-second aspect, the present disclosure provides a method for the isolation of the circulating, cell-free nucleic acid, the method comprising subjecting a sample containing the cell free complex to cesium-chloride (CsCl) density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the circulating, cell-free nucleic acid from the gradient. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-third aspect, the present disclosure provides a method for the detection of circulating, cell-free nucleic acid, the method comprising isolating the circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the circulating, cell-free nucleic acid and detecting the circulating, cell-free nucleic acid. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and detecting the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-fourth aspect, the present disclosure provides a method for analysis of circulating, cell-free nucleic acid, the method comprising isolating the circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the circulating, cell-free nucleic acid and analyzing the circulating, cell-free nucleic acid to determine a characteristic of the nucleic acid. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In an twenty-fifth aspect, the present disclosure provides a method for analysis of circulating, cell-free nucleic acid, the method isolating the circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the circulating, cell-free nucleic acid and analyzing the circulating, cell-free nucleic acid to determine the presence of a mutation, the presence of a polymorphism, the methylation state, the concentration of a nucleic acid, the level of expression of a nucleic acid or a combination of the foregoing. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-sixth aspect, the present disclosure provides a method for determining a biologically relevant nucleic acid profile of a subject, the method comprising isolating circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the circulating, cell-free nucleic acid and identifying a plurality of nucleic acids to produce the nucleic acid profile. Such method may further comprise comparing the subject profile to a corresponding nucleic acid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding nucleic acid profile indicative of a disease state. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-seventh aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the cell-free nucleic acid and determining the presence of a nucleic acid characteristic, wherein the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In a twenty-eight aspect, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising generating a nucleic acid profile from a subject as set forth in the twenty-sixth aspect and comparing the generated profile to a corresponding disease fingerprint and determining the subject is suffering from or at risk for a particular disease if the subject profile contains one or more characteristics of the disease fingerprint. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA

DETAILED DESCRIPTION
Definitions

As used herein the term “cell free complex” refers to a complex comprising a cell free nucleic acid component and at least one cellular component. Such cellular components include but are not limited to, polypeptides, proteins and lipids. The cell free complex may contain a nucleic acid component, a polypeptide component and a lipid component or a nucleic acid component and one of a polypeptide component and a lipid component. For clarity, while the term “cell free complex” requires the presence of a nucleic acid component and at least one cellular component, the term also includes nucleic acids that are not associated with a cellular component provided that at least a portion of the nucleic acids in the cell free complex are associated with at least one additional cellular component.

As used herein, the term “genomic characteristic” or “nucleic acid characteristic” refers to a characteristic of the nucleic acid contained in the cell free complex. Any characteristic that is used to provide information regarding the nucleic acid may be used. Such characteristics include, but are not limited to, the presence of mutations, the presence of polymorphisms, the methylation pattern, the concentration, the expression level and the profile of nucleic acids contained in the cell free complex or associated with the cell-free nucleic acid. The foregoing characteristics may be determined by comparing to a wild-type counterpart of the nucleic acid.

As used herein, the term “proteomic characteristic” or “polypeptide characteristic” refers to a characteristic of the polypeptides contained in the cell free complex. Any characteristic that is used to provide information regarding the polypeptide may be used. Such characteristics include, but are not limited to, the presence of mutations, the presence of post-translational modifications (for example, phosphorylation), the presence of insertions or deletions, the concentration, the expression level and the profile of polypeptides contained in the cell free complex or associated with the cell-free nucleic acid. The foregoing characteristics may be determined by comparing to a wild-type counterpart of the polypeptide.

As used herein, the term “lipidomic characteristic” or “lipid characteristic” refers to a characteristic of the lipids contained in the cell free complex. Any characteristic that is used to provide information regarding the lipid may be used. Such characteristics include, but are not limited to, the presence of altered forms (for example, carbon chain length), the presence of modifications (for example, changes in numbers of double and/or triple bonds), the concentration, the expression level and the profile of lipids contained in the cell free complex or associated with the cell-free nucleic acid. The foregoing characteristics may be determined by comparing to a wild-type counterpart of the lipid.

As used herein, the term “isolated” means the partial purification or increase in concentration of a component of a sample, such as the cell free complex or a component thereof. Such isolation does not require absolute purification. For example, when isolated is used in reference to a nucleic acid, the nucleic acid may comprise 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of 99% or more of the material (by weight) in the mixture isolated.

As used herein, the term “subject” may be any mammal and in one embodiment is a human. The human subject may be any age. The human subject may also be an unborn child.

Introduction

As discussed above, the present methods for isolating circulating, cell free nucleic acid suffer from several disadvantages. The present disclosure overcomes these disadvantages by providing novel centrifugation methods for the isolation of circulating, cell free nucleic acid and/or a cell free complex comprising a circulating, cell free nucleic acid component along with associated cellular components. When a cell free complex is isolated, the nucleic acid component of the complex, in one embodiment, is associated with cellular components, such as, but not limited to, polypeptides and lipids. In certain embodiment, the circulating cell free nucleic acid is associated with both polypeptides and lipids. Such association may be direct or indirect. However, the nucleic acid is not required to be physically associated with the cellular components. For example, the nucleic acid component may simply be located in the same density gradient as the cellular components. Furthermore, in certain embodiment, the circulating cell free nucleic acid may not be associated with a cellular component. The methods of the present disclosure provide for the isolation of circulating cell free nucleic acids that are longer and less fragmented as compared to prior art methods. In one embodiment, such a result is due, at least in part, to protection from degradation of the nucleic acid component by the associated cellular components. Furthermore, the circulating, cell free nucleic acid represent biologically relevant nucleic acid targets as the methods described selectively remove non-functional nucleic acids from the isolated material. The nucleic acid component of the isolated complexes reflects a wide spectrum of genomic representation enabling successful detection of mutations, polymorphisms, methylation status and other genomic markers for use in diagnostic and therapeutic applications. Furthermore, the additional cellular components in the cell free complexes isolated provide information regarding the proteomic and lipidomic status of the subject. Such markers are also useful in diagnostic and therapeutic applications. The resulting isolated cell free complex therefore provides a rich source of information regarding the genomic, proteomic and lipidomic status of a subject.

In principle, such centrifugation approaches requires no prior information about the cell free complexes, including the identity or composition of the nucleic acid, protein and lipid components, and is very sensitive since little starting material is needed. Most importantly, taking into account the fact that the vast majority of human genome doesn't code for proteins (over 98%), the selective enrichment provided by the methods of the present disclosure are superior to the methods of the prior art which rely on blind extraction methods that can lose up to 80% of circulating, cell free nucleic acids during extraction.

While not wishing to be bound by any particular theory, certain regions of nucleic acid (for example, enhancers, transcribed exons, or active promoters) are associated with cellular components during normal function. As a result, such nucleic acids have a density that is intermediate between free nucleic acid and protein. Therefore, density gradient centrifugation methods may be used to preferentially isolated cell free complexes that contain a nucleic acid component that is associated with additional cellular components.

Methods of Isolating Cell Free Complexes and Components Therein

The methods of the present disclosure provide for the isolation of cell free complexes. Such cell free complexes comprise a cell free nucleic acid and at least one cellular component. Such cellular components include but are not limited to, polypeptides, proteins and lipids. The circulating, cell-free nucleic acids and cell free complexes isolated provide a biologically relevant, information rich source of information regarding the subject.

In one embodiment, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to centrifugation and isolating the cell free complex. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid.

In one embodiment, the present disclosure provides a method for the isolation of a cell free complex circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to density gradient centrifugation and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex.

In one embodiment, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex.

In one embodiment, the present disclosure provides a method for the isolation of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising subjecting a sample containing the cell free complex to cesium-chloride (CsCl) density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the cell free complex from the gradient. Such method may further comprise isolating at least one cell-free nucleic acid from the complex, the nucleic acid optionally associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid. Such method may further comprise isolating at least cellular component from the complex.

In a particular embodiment of the foregoing, the density gradient is formed from CsCl. In a further particular embodiment, the CsCl density gradient has a density of between 1.30 and 1.45 g/cm³, 1.35 to 1.40 g/cm³, 1.35 to 1.45 g/cm³, 1.38 to 1.43 g/cm³or 1.39 to 1.42 g/cm³. In a further particular embodiment, the nucleic acid is DNA, RNA or a combination of both DNA and RNA. Any form of known form of nucleic acid is included in the definition of nucleic acid.

In a particular embodiment, the length of the nucleic acid fragment isolated in the cell free complexes is over 500, over 750, over 1000, over 2000 or over 50000 base pairs. In another embodiment, the lent hog the nucleic acid fragments in the cell free complex is between 500 and 5000 base pairs, 1000 to 10,000 base pairs, 1000 to 5000 base pairs, or 10,000 to 20,000 base pairs.

In a particular embodiment of the foregoing, the nucleic acid is DNA, RNA or a combination of the foregoing. In a further particular embodiment, the nucleic acid is DNA. In a further particular embodiment, the nucleic acid is RNA.

In a particular embodiment of the foregoing, the associated cellular component is a lipid, a protein, a polypeptide or a combination of the foregoing. In a further particular embodiment, the cellular component is a lipid. In a further particular embodiment, the cellular component is a protein. In a further particular embodiment, the cellular component is a polypeptide.

In a particular embodiment of the foregoing, the cell free complex may be subject to additional processing steps, for example, to further isolate the cell-free nucleic acid and/or the cellular components from the cell free complex. Any means known in the art to accomplish such ends is included in the scope of the present disclosure.

Methods of Isolating Circulating, Cell-Free Nucleic Acid

The methods of the present disclosure provide for the isolation of circulating, cell-free nucleic acids. Such nucleic acid may be DNA and/or RNA. The circulating, cell-free nucleic acids provide a biologically relevant, information rich source of information regarding the subject.

In one embodiment, the present disclosure provides a method for the isolation of a circulating, cell-free nucleic acid comprising subjecting a sample containing the circulating, cell-free nucleic acid to centrifugation and isolating the circulating, cell-free nucleic acid.

In another embodiment, the present disclosure provides a method for the isolation of a circulating, cell-free nucleic acid comprising subjecting a sample containing the circulating, cell-free nucleic acid to density gradient centrifugation and isolating the circulating, cell-free nucleic acid from the gradient.

In another embodiment, the present disclosure provides a method for the isolation of a circulating, cell-free nucleic acid comprising subjecting a sample containing the circulating, cell free nucleic acid to density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the circulating, cell free nucleic acid from the gradient.

In another embodiment, the present disclosure provides a method for the isolation of a circulating, cell-free nucleic acid comprising subjecting a sample containing the cell free complex to cesium-chloride (CsCl) density gradient centrifugation with a buoyant density of 1.3 to 1.45 g/cm³and isolating the circulating, cell-free nucleic acid from the gradient.

In a particular embodiment of the foregoing, the circulating, cell-free nucleic acid may be present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In a further particular embodiment of the foregoing, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid. In one instance, the cellular component is a lipid or a protein.

In a particular embodiment of the foregoing, the circulating cell free nucleic acid may be subject to additional processing steps, for example, to further isolate the cell-free nucleic acid from a cell free complex or associated cellular components. Any means known in the art to accomplish such ends is included in the scope of the present disclosure.

Methods of Detection and Analysis of Cell Free Complexes

The present disclosure also provides methods for the detection and analysis of the circulating, cell-free nucleic acid and the components of the cell free complex isolated as described herein.

In one embodiment, the present disclosure provides a method for the detection of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any one of the first through fourth aspects, optionally further processing the cell free complex and associated cellular components and detecting the cell-free nucleic acid, the associated cellular component or a combination of the foregoing.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the cell-free nucleic acid, associated cellular components or a combination of the foregoing to determine a characteristic of the nucleic acid, associated cellular components or a combination of the foregoing.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the associated polypeptide component to determine a polypeptide characteristic. The method may further comprise analyzing a nucleic acid component and/or an additional cellular component.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the associated lipid component to determine a lipid characteristic. The method may further comprise analyzing a nucleic acid component and/or an additional cellular component.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the cell-free nucleic acid to determine the presence of a mutation, the presence of a polymorphism, the methylation state, the concentration of a nucleic acid, the level of expression of a nucleic acid, the nucleic acid profile or a combination of the foregoing. The method may further comprise analyzing a cellular component.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the polypeptide component to determine the presence of a mutation, the presence of a post-translational modification, the presence of insertions or deletions, the concentration, level of expression of a nucleic acid, the profile of the polypeptides or a combination of the foregoing. The method may further comprise analyzing an additional cellular component or a nucleic acid.

In one embodiment, the present disclosure provides a method for analysis of a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components, the method comprising isolating the cell free complex as set forth in any of the methods described herein, optionally further processing the cell free complex and analyzing the lipid component to determine the presence of altered forms, the presence of modifications, the concentration, the expression level, the profile of lipids or a combination of the foregoing.

Methods of Detection and Analysis of Circulating Cell-Free Nucleic Acid

The present disclosure also provides methods for the detection and analysis of circulating, cell-free nucleic acid.

In one embodiment, the present disclosure provides a method for analysis of circulating, cell-free nucleic acid, the method comprising isolating the circulating, cell-free nucleic acid as set forth in any of the methods described herein, optionally further processing the circulating, cell-free nucleic acid and analyzing the circulating, cell-free nucleic acid to determine a characteristic of the nucleic acid. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing.

In one embodiment, the present disclosure provides a method for analysis of circulating, cell-free nucleic acid, the method comprising isolating the circulating, cell-free nucleic acid as set forth in any of the methods described herein, optionally further processing the circulating, cell-free nucleic acid and analyzing the circulating, cell-free nucleic acid to determine the presence of a mutation, the presence of a polymorphism, the methylation state, the concentration of a nucleic acid, the level of expression of a nucleic acid or a combination of the foregoing

In a particular embodiment of the foregoing, the circulating, cell-free nucleic acid may be present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In a further particular embodiment of the foregoing, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the at least one cellular component. In one instance, the cellular component is a lipid or a protein.

Biologic Profiles

The present disclosure also allows the generation of biologically relevant profiles of the cell-free nucleic acid, polypeptides and/or lipids isolated as described herein. In one embodiment, the profile comprises one or more nucleic acid, polypeptide or lipid characteristics. In another embodiment, the profile comprises one or more nucleic acids, polypeptides or lipids isolated as described herein. Such biological profiles may be used for various purposes, including but not limited to, monitoring the health of a subject over time and for use in methods of diagnosis or treatment.

In one embodiment, the present disclosure provides a method for determining a biologically relevant nucleic acid, polypeptide or lipid profile of a subject, the method comprising isolating a cell free complex comprising circulating cell free nucleic acid and associated cellular components, optionally further processing the complex and identifying one or more nucleic acids, polypeptides and/or lipids present in the complex. Such method may further comprise comparing the subject profile to a corresponding profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding profile indicative of a disease state. Such corresponding profile may be a disease fingerprint as described herein.

In one embodiment, the present disclosure provides a method for determining a biologically relevant nucleic acid, polypeptide or lipid profile of a subject, the method comprising isolating a cell free complex comprising circulating cell free nucleic acid and associated cellular components, optionally further processing the complex and identifying one or more nucleic acid, polypeptide or lipid characteristics of the nucleic acids, polypeptides and/or lipids present in the complex. Such method may further comprise comparing the subject profile to a corresponding profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding profile indicative of a disease state. Such corresponding profile may be a disease fingerprint as described herein.

In one embodiment, the present disclosure provides a method for determining a biologically relevant nucleic acid profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one the methods described herein, optionally further processing the cell free complex and identifying a plurality of cell-free nucleic acids or one or more nucleic acid characteristics to produce the nucleic acid profile. Such method may further comprise comparing the subject profile to a corresponding nucleic acid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding nucleic acid profile indicative of a disease state. Such corresponding profile may be a disease fingerprint as described herein.

In one embodiment, the present disclosure provides a method for determining a biologically relevant polypeptide profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one the methods described herein, optionally further processing the cell free complex and identifying a plurality of polypeptides or one or more polypeptide characteristics to produce the polypeptide profile. Such method may further comprise comparing the subject profile to a corresponding polypeptide profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding polypeptide profile indicative of a disease state. Such corresponding profile may be a disease fingerprint as described herein.

In one embodiment, the present disclosure provides a method for determining a biologically relevant lipid profile of a subject, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any one the methods described herein, optionally further processing the cell free complex and identifying a of lipids or one or more lipid characteristics to produce the lipid profile. Such method may further comprise comparing the subject profile to a corresponding lipid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding lipid profile indicative of a disease state. Such corresponding profile may be a disease fingerprint as described herein.

In one embodiment, the present disclosure provides a method for determining a biologically relevant nucleic acid profile of a subject, the method comprising isolating circulating, cell-free nucleic acid as set forth in any one of the nineteenth to twenty-second aspects, optionally further processing the circulating, cell-free nucleic acid and identifying a plurality of nucleic acids to produce the nucleic acid profile. Such method may further comprise comparing the subject profile to a corresponding nucleic acid profile indicative of a disease state and determining the subject is suffering from or at risk for a particular disease or condition if the subject profile contains one or more characteristics of the corresponding nucleic acid profile indicative of a disease state. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA. Such corresponding profile may be a disease fingerprint as described herein.

In another embodiment, the present disclosure provides for a method of determining a disease fingerprint of a particular disease or condition, the method comprising isolating a cell free complex comprising circulating cell free nucleic acid from a subject known to have a particular disease or condition (or known/deemed to be healthy or not suffering from the disease or condition), optionally further processing the cell free complex, generating a nucleic acid, polypeptide or lipid profile of the disease or condition, generating a corresponding nucleic acid, polypeptide or lipid profile from a subject that is not suffering from a particular disease or condition, and comparing the disease and control profiles to identify nucleic acid, polypeptide and/or lipid characteristics in the disease profile that are absent in the control profile or that are present in the control profile, but absent in the disease profile. Such characteristics may be referred to as a disease fingerprint and compared to the corresponding profiles generated from subjects according to the methods of the present disclosure

Methods of Diagnosis

Through the use of the isolated cell free complexes of the present disclosure, it can be determined if a subject is suffering from or at risk for a disease or condition. The disease or condition may be any disease or condition known in the art, such as for example, cancer, heart disease, or diseases and conditions associated with genetic or protein abnormalities.

In one embodiment, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components or circulating cell-free nucleic acid as set forth in any one of the methods described herein, optionally further processing the cell free complex and determining the presence of a characteristic associated with the disease in the components of the complex. Such characteristic may be the presence of nucleic acid characteristic, a proteomic characteristic or a lipid characteristic.

In another embodiment, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components or circulating cell-free nucleic acid as set forth in any one of the methods described herein, optionally further processing the cell free complex and determining the presence of a nucleic acid characteristic in the cell-free nucleic acid. In one embodiment the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing.

In another embodiment, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating a cell free complex comprising circulating, cell-free nucleic acid and associated cellular components as set forth in any of the methods described herein, optionally further processing the cell free complex and determining the presence of a polypeptide and/or lipid characteristic in the cellular component. In one embodiment, the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such polypeptide characteristic includes, but is not limited to, a mutation, the presence of post-translational modifications, the presence of insertions or deletions, the concentration, level of expression of a nucleic acid, the profile of the polypeptides or a combination of the foregoing. Such lipid characteristic includes, but is not limited to, the presence of altered forms, the presence of modifications, the concentration, the expression level, the profile of lipids or a combination of the foregoing.

In another embodiment, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising isolating circulating, cell-free nucleic acid as set forth in any one of methods described herein, optionally further processing the cell-free nucleic acid and determining the presence of a nucleic acid characteristic. In one embodiment, the subject is judged to be suffering from or at risk for a disease if the presence of any of the characteristic is associated with the disease. Such nucleic acid characteristic includes, but is not limited to, a mutation, a polymorphism, methylation state, concentration of a nucleic acid, level of expression of a nucleic acid, the profile of the nucleic acids or a combination of the foregoing. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA.

In another embodiment, the present disclosure provides a method for determining if a subject is suffering from or at risk for a disease, the method comprising generating a nucleic acid profile from a subject as set forth in any of the methods described herein and comparing the generated profile to a corresponding disease fingerprint and determining the subject is suffering from or at risk for a particular disease if the subject profile contains one or more characteristics of the disease fingerprint. In one embodiment of this aspect, the circulating, cell-free nucleic acid is present in a cell free complex and such method may further comprise isolating at least one cell-free nucleic acid from the complex. In another embodiment, the circulating, cell-free nucleic acid is associated with a cellular component, such as, but not limited to, a polypeptide and/or a lipid, and such method may further comprise isolating at least one cell-free nucleic acid from the cellular components. Such method may further comprise isolating at least one cellular component from the circulating, cell-free nucleic acid and analyzing the cellular component. In one instance, the cellular component is a lipid or a protein. Such nucleic acid may be DNA and/or RNA

In any of the foregoing, the circulating, cell-free nucleic acid and cell free complex may be isolated by any method described herein. The determined characteristic, whether a nucleic acid, polypeptide or lipid characteristic, may inform a healthcare provider if the subject is suffering from or at risk for a particular disease or condition. For example, if a nucleic acid characteristic is analyzed, such as the presence of a mutation disposing an individual to a cancer risk, the individual may be monitored for the increased risk, undergo additional testing or initiate lifestyle changes or a combination of the foregoing.

Furthermore, in the foregoing embodiment, the determined characteristic, whether a nucleic acid, polypeptide or lipid characteristic may be compared to a control to determine if the subject is at risk for a particular disease or condition. For example, a nucleic acid profile of the nucleic acid components in the cell free complex may be prepared as described herein. The nucleic acid profile may be compared to a control nucleic acid profile obtained under the same or similar conditions that is free of a particular disease. Furthermore, profiles may be taken from individuals known to have a particular disease or condition, and these profiles compared to a control profile to identify nucleic acids that are subject to increased or decreased concentration to generate a nucleic acid fingerprint of a particular disease or condition comprising such nucleic acids that undergo changes in concentration. The presence of one or more such nucleic acids in a subject nucleic acid profile may indicate that the subject is suffering from or at risk for the disease or condition. The same analysis may be undertaken for the polypeptide components and the lipid components of the cell free complexes.

Determining Methods of Treatment

The present disclosure also provides for determining a beneficial or optimal course of treatment for an individual. Such treatment decisions can be of use to a healthcare provider when choosing between various forms of treatment, especially when physiological feature of the subject may impact the efficacy of such treatment or inform the healthcare provider on the dose of a particular medication to be used in such treatment.

In one embodiment, the present disclosure provides a method for determining beneficial or optimal course of treatment, the method comprising isolating a cell free complex comprising circulating cell free nucleic acid and associated cellular components or circulating cell-free nucleic acid, optionally further processing the cell free complex and determining the presence of a characteristic that may impact a particular course of treatment and modifying the course of treatment if the characteristic is present. Such characteristic may be the presence of genomic characteristic, a proteomic characteristic or a lipidomic characteristic.

For example, consider planning a course of treatment using the drug Plavix. Plavix is used to prevent blood clots after a recent heart attack or stroke, and in people with certain disorders of the heart or blood vessels. The effectiveness of Plavix in some individuals is dependent on the metabolism of the drug by certain liver enzymes, particularly CYP2C19. Metabolism is required for optimal effectiveness of the drug. Certain polymorphisms are indicative of those individuals that do not metabolize Plavix effectively and as a result may not receive the full benefits of the drug at standard dosing. By identifying such polymorphisms, a healthcare provider may plan the most beneficial or optimal course of treatment by prescribing another drug or adjusting the administered dose of Plavix.

Cell Free Complexes

The present disclosure provides methods for the isolation of cell free complexes comprising a cell free nucleic acid component and at least one cellular component. The at least one cellular component may be associated with a nucleic acid directly, or indirectly (such as through interaction with another cellular component). Such additional cellular components include but are not limited to, polypeptides, proteins and lipids. The presence of the at least one cellular component may aid in the preservation of the nucleic acid component, such as by protecting the nucleic acid component from degradation. As a result, nucleic acids of increased molecular weight are isolated by the methods of the present disclosure as compared to prior art methods. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the nucleic acids in the cell free complex are associated with at least one additional cellular component.

The cell free complex isolated as disclosed herein may be analyzed as is known by one of ordinary skill in the art. For example, the nucleic acid component may be analyzed by any known genomic methods to determine a genomic characteristic, the polypeptide component may be analyzed by any known proteomic methods to determine a proteomic characteristic and the lipid component may be analyzed by any known lipidomic methods to determine a lipidomic characteristic. As such, information regarding the status of the subject may be obtained and used for diagnostic, prognostic or therapeutic applications.

Samples

In one embodiment of the above method, a sample is obtained from the patient. Any type of sample routinely used in the art may be used, such as but not limited to, blood, plasma, serum, bone marrow, urine, amniotic fluid, placental blood, buccal swabs or other sample types. In one embodiment, the sample is a blood sample or plasma obtained from the patient. The blood sample may be collected and processed as is known in the art. In one embodiment, the blood samples are collected in an anti-chelating agent such as EDTA or another calcium binding agent. The blood sample may be subject to purification, such as, but not limited to, centrifugation, in order to separate the plasma fraction from other components of the blood. In one embodiment, the sample is a plasma sample. The sample may be stored at 4 degrees Celsius or lower prior to the purification step if desired. The sample may be stored cryogenically after purification for future analysis. In one embodiment, the sample is may be processed to remove cellular components, such as blood cells. In one embodiment, the sample is not subject to steps to purify or concentrate nucleic acid or another cellular component contained in the sample, with the understanding that procedures to separate plasma from blood and similar processing steps are not considered to be steps that purify or concentration nucleic acid or another cellular component contained in the sample.

Isolation of Cell Free Complexes and Circulating Cell-Free Nucleic Acid

The circulating cell-free nucleic acid and cell free complexes are separated using centrifugation. The embodiments described herein are useful in isolating both circulating cell-free nucleic acid and cell free complexes. In certain embodiments, the methods isolate both circulating cell-free nucleic acid and cell free complexes. In one embodiment, density gradient centrifugation is used. In a particular embodiment vertical spin density gradient ultracentrifugation is used. However, any density gradient separation means known in the art may be used.

In a typical samples, using density gradient centrifugation, the circulating cell-free nucleic acid and/or cell free complexes are separated from other components in the sample. For a typical sample containing free nucleic acid, nucleic acid complexes and polypeptides/proteins, the foregoing are separated in the following order (from the bottom of the density gradient to the top of the density gradient): polypeptide/proteins, circulating cell-free nucleic acid (which may be associated with cellular components) and cell free complexes as described herein and free nucleic acid. A variety of density gradient centrifugation conditions may be used. The composition of the density gradient may impact the separation between various components in the sample. The following are illustrated by way of example only and should not be interpreted as limiting the scope of the separation techniques to density gradient centrifugation or as limiting the conditions employed in density gradient centrifugation to those conditions specified.

As discussed above, in some cases certain a given sample may contain more than one type of component. Specific density gradient centrifugation conditions may be employed to provide maximum resolution of the various components in a sample or may be employed to provide maximum resolution of a selected component in the sample, such as the cell free complexes described herein.

In one embodiment, the density gradient centrifugation conditions and parameters are selected to provide maximum resolution of the circulating cell-free nucleic acid and/or cell free complexes described herein. Conditions and parameters that may be varied include, but are not limited to, density of the gradient, density of the layers comprising the density gradient, volume of the layers comprising the density gradient, centrifugation time settings, acceleration setting (impacting the time it takes for the centrifuge to reach a set RPM), deceleration settings (impacting the time it takes for the centrifuge to come to a stop from the set RPM at the end a specified time setting), speed of the centrifuge (measured in RMP), centrifugal force applied to the sample (measured in x g) and temperature of the centrifugation run. The various parameters discussed above may be varied singly or in any combination desired.

In one embodiment, the density gradient comprises a formed gradient of uniform composition. In another embodiment, the density gradient comprises two layers of gradient material (referred to as a top and bottom layer). A commonly used density gradient material is CsCl. Other commonly used density gradient materials include KBr, sucrose, and colloidal silica particles coated with polyvinylpyrrolidone (such as the product sold as Percoll®). Any density gradient solution known in the art to create the required density range may be used. In one embodiment, the density gradient is formed from CsCl. Centrifugation will be performed in an appropriate vessel, such as a centrifuge tube. A variety of suitable centrifuge tubes are commercially available, for example from Beckman-Coulter, of Brea, Calif. In a specific embodiment separation is achieved using a single spin.

In one embodiment, the density of the gradient is from 1.30 to 1.45 g/cm³. In another embodiment, the density of the gradient is from 1.30 to 1.40 g/cm³. In another embodiment, the density of the gradient is from 1.35 to 1.40 g/cm³. In another embodiment, the density of the gradient is from 1.35 to 1.45 g/cm³. In another embodiment, the density of the gradient is from 1.38 to 1.43 g/cm³. In another embodiment, the density of the gradient is from 1.39 to 1.42 g/cm³. In another embodiment, the density of the gradient is from 1.35 to 1.42 g/cm³. In one embodiment, the volume of the density gradient is from 3-5 mls. In one embodiment, the volume of the density gradient is 5.0 mls.

In one embodiment where two layers of density gradient material are used, the bottom layer ranges from 1.10 to 1.40 g/cm³, from 1.15 to 1.30 g/cm³or from 1.15 to 1.25 g/cm³and the density of the top layer ranges from 0.5 to 1.2 g/cm³, from 1.0 to 1.15 g/cm³or from 1.0 to 1.10 g/cm³. In another aspect of this embodiment, the density of the bottom layer is 1.21 g/ml or 1.30 g/cm³and the density of the top layer is 1.05 g/cm³. Further, in one aspect of this embodiment, the volume of the bottom layer ranges from 0.2 to 4.0 ml, from 0.8 to 2.5 ml or from 1 to 2 ml and the volume of the top layer ranges from 1 to 4.8 ml, from 1.2 ml to 3.0 ml or from 3.0 to 4.0 ml. In another aspect of this embodiment, the volume of the bottom layer is 2.0 ml or 1.0 ml and the volume of the top layer is 2.90 ml or 3.9 ml.

Further, in one aspect of this embodiment, the settings for the ultracentrifuge are varied as follow: (i) centrifugation time from 1 to 4 hours (note that centrifugation time does not include the time required for deceleration of the centrifuge rotor), from 1 to 3 hours or from 2 to 4 hours; (ii) centrifugation speed from 190,000×g to 555,000×g or 275,000×g to 480,000×g; and (iii) centrifugation temperature from 15 to 30 degrees Celsius or from 20 to 25 degrees Celsius. Furthermore, in one aspect of this embodiment, the acceleration and deceleration settings are selected provide appropriate acceleration and deceleration profiles in order to maximize the desired separation. In one aspect, the acceleration and/or deceleration phases of the spin are set to be slow in order to minimize vibrations that may occur during a quick acceleration and/or deceleration. In one aspect, the acceleration and/or deceleration phases of the spin are set to be fast in order to resolve a given class of lipoprotein. For example, using a Beckman Coulter ultracentrifuge (Optima™ XL-100 K Ultracentrifuge), the acceleration and/or deceleration settings may range from 5 to 9 or 8 to 9 (with 9 being the slowest setting). In another aspect of this embodiment, the acceleration and/or deceleration settings may range from 1 to 5 or 2 to 4 (with 1 being the fastest setting).

In one embodiment, the following conditions are used: (i) density gradient of 1.30 to 1.45 g/cm3; (ii) 3 hour spin time; (iii) 20 degrees Celsius; (iv) 416,000×g and (v) minimum acceleration/deceleration settings. TIMES G

Use of Comparative Database

As disclosed herein, the results of analysis of the components of the cell free complex, including cell-free nucleic acid, obtained as described herein may be compared with a standard or control. For example, such a control may be a sample from a subject who is determined to have or be free of a given disease or condition. Furthermore, the control may be a compilation of the results obtained from a population of individuals who are determined to have or be free of a given disease or condition. In such a case, the results may be contained within a comparative database.

When a comparative database is used as a control, the comparative database may be constructed in a variety of ways. Furthermore, the individuals in the comparative database may be matched to the subject being tested based on a stratification criterion or may be non-matched as compared to the subject. In one embodiment, the stratification criterion is age. In another embodiment, the stratification criterion is ethnic origin. For example, if the subject is 65 years of age, in one embodiment the comparative database may be composed of individuals with ages from, for example, 60 to 70 years, or in a second embodiment, the comparative database may be composed of individuals with ages from, for example, 25 to 40 years. The use of a comparative database comprising a younger population may offer certain advantages since the younger subjects that comprise the population will be more likely to be free of disease states and other conditions that may impact the analysis. Using an age matched population for the comparison may actually decrease the sensitivity of the method since the age matched population of the comparative database may in fact have a certain risk for the disease or condition being analyzed.

The individuals making up the comparative database may be healthy (i.e., disease free) or they may be selected based on their diagnosis of a particular disease or condition, or both. If healthy individuals are selected, the characteristic determined from the subject, such as the presence of a particular mutation or polymorphism or the nucleic acid and/or polypeptide profile, can be compared with the corresponding characteristic from healthy individuals in the comparative database. If individuals with a diagnosed disease state are selected, the characteristic determined from the subject, such as the presence of a particular mutation or polymorphism or the nucleic acid and/or polypeptide profile, can be compared with the corresponding characteristic from individuals diagnosed with a disease states and/or defined stages of a disease state in the comparative database. In this manner, the comparison may be able to predict if a subject is at risk for a particular disease or condition (from a comparison with healthy individuals in the comparative database), if the subject is suffering from a particular disease or condition (from a comparison with individuals in the comparative database diagnosed with such disease or condition) or to diagnose severity (from a comparison with individuals in the comparative database diagnosed with various stages of a disease or condition). The stratification of the database, as discussed below, may aid in making such comparisons.

The comparative database may be stratified based on a number of stratification criteria. These criteria may be risk factors, demographic factors, other relevant factors or a combination of the preceding. Demographic factors include, but are not limited to, age, gender and ethnicity. The inclusion of a specific stratification criteria as a risk factor or demographic factor may be modified (for example, age may be considered both a risk factor and a demographic factor). The individuals in the comparative database may be tagged or otherwise identified, such that the appropriate population of individuals in the comparative database may be selected for the comparison to the subject.

Furthermore, the comparative database may be refined over time. The individuals in the database may be followed over time and their health status monitored. If an individual no longer meets an inclusion criterion for the comparative database, the individual may be removed or their information modified. In this manner the quality of the comparative database may be improved over time, resulting in a database with improved sensitivity and specificity.

The characteristic determined from the subject, such as the presence of a particular mutation or polymorphism or the nucleic acid, lipid and/or polypeptide profile, may then be compared to the corresponding characteristic from appropriately selected defined group of individuals in a comparative database. Appropriately selected means that the selected characteristic from a defined group of individuals in the comparative database is selected for comparison to the selected characteristic determined for the subject. The defined group may be all the individuals in the comparative database or less than all the individuals in the comparative database. The defined group may be selected on the basis of stratification criteria as discussed above. The healthcare provider may select the defined group, with such selection based on one or more defining characteristics of the subject. In one embodiment, the defined group may be selected on the basis of ethnicity (African-American), gender (male), health status (disease free or diagnosed with a particular disease or condition), and age (20-45 years of age or 55-65 years of age). Furthermore, the comparison may be carried out multiple times for any given subject to various iterations of the comparative database. For example, given a 60 year old, non-smoking, African-American male subject, comparisons could be made using a defined group from the database selected on the basis of gender (male) only, gender and age, or selected to include all individuals in the comparative database.

Results
Example 1—General Procedures
Sample Handling

Blood samples were collected in 10 ml lavender top EDTA collection tubes using standard phlebotomy practices. Immediately after collection, the tubes were gently inverted 4-6 times and then centrifuged at 2,500 RPM for 15 minutes at 4° C. The supernatant plasma was transferred into 2 ml cryogenic vials and frozen at −80° C. until analysis. No further modification of manipulation of the blood sample is required. The supernatant plasma is used directly in the methods as described without further amplification of purification of any component of the plasma, such as, but not limited to, nucleic acids and polypeptides.

VAP Analysis

VAP cholesterol patent tests were performed at Atherotech Diagnostic Laboratories (Birmingham, Ala.) according to internal standard operating procedures. The VAP method separates lipoproteins based on their density using single vertical-spin density gradient ultracentrifugation, then quantifies cholesterol content (Kulkarni, et al., Clin Lab Methods, 26, 787-802, 2006; Kulkarni et al., J. Lipid Res, 35, 159-168, 1994; Kulkarni et al., J Lipid Res, 38, 2353, 2364, 1997).

Density Gradient Centrifugation

Twenty μl of plasma was diluted in 980 μl of 1× phosphate buffered saline (PBS) prior to mixing with a CsCl gradient solution. The CsCl gradient was prepared by adding 2.19 g of CsCl to 4 ml of 1× Tris-EDTA buffer; sample volume was adjusted to 5 ml to a final density of 1.30 to 1.45 g/cm3. The resulting sample was centrifuged using a Beckman Coulter Optima XL-100K ultracentrifuge and Beckman VTI 65.2 rotor 3 hours at 65,000 RPM at 20° C. (416,000×g). 250 μl fractions (fractions 1-20) were collected with a peristalitic pump. Detection and quantification of the cell free nucleic acid was conducted by measuring uv absorbance at 260 nm by a NanoDrop ND-8000 spectrophotometer (ThermoScientific).

For further analysis of the nucleic acid, polypeptide and lipid components, the fractions were desalted and concentrated on a Microcon DNA fast flow centrifugal filter column. Nucleoprotein complexes in the solution were disassociated by alkaline denaturation. Free nucleic acid complexes were subject to isothermal amplification to amplify cell free DNA acid or reverse transcription on cell free RNA. Subsequent analysis was carried out using commercially available kits according to manufacturers' instructions.

Example 2—Overview of General Procedure

FIG. 1 shows an overview of one embodiment of the general procedure of the methods as disclosed herein. As shown in FIG. 1, a sample (in this example, a blood sample processed as described above). The sample is added to an appropriate vessel for density gradient centrifugation. Any suitable vessel may be used as is known in the art. In this example, a Beckman Coulter OptiSeal tube is used. The sample is then subject to density gradient centrifugation as described herein. While a number of density gradient centrifugation procedures may be used as is known in the art (including those described herein), in many of the examples described herein the density gradient is a CsCl density gradient to yield a final density of 1.3 to 1.45 g/cm³. The centrifugation protocols may also be varied as is known in the art (including those protocols described herein. In many of the examples described herein, the centrifugation conditions are as follow: 3 hour spin at 65,000 rpm at 20° C. using a Beckman Coulter Optima XL-100K ultracentrifuge and Beckman VTI 65.2 rotor (416,000×g).

At the end of the centrifugation procedure, the vessel containing the sample is gently removed and the bottom of the tube is punctured to allow removel of the sample from the bottom of the tube. Fractions are collected using a peristaltic pump for analysis. Using this method, the bulk nucleic acid (containing less or no associated cellular components) is lower in the density gradient and is collected first (generally having a density around 1.70 g/cm³), followed by the cell free nucleic acid associated with cellular components and cell free complexes (generally having a density around 1.30 to 1.45 g/cm³), followed by a protein fraction (generally having a density around 1.25 g/cm³). Any convenient aliquot volume may be used, for example 250 μl fractions. The collected fractions are subject to ultraviolet absorbance measurement at 260 nm using a spectrophotometer (such as a NanoDrop ND-8000) to detect and quantify the nucleic acid and other components present in the collected fractions.

The fractions may then be further processed as is known in the art for subsequent analysis. For example, for genomic analysis the fractions may be deslated and concentrated using methods known in the art (for example, a Microcon DNA Fast Flow centrifugal filter column). Nucleoprotein complexes may be dissociated using standard techniques (such as alkaline denaturation). Other processing steps may also be performed as is known in the art. In one embodiment, such further processing steps do not involve the purification of nucleic acid isolated. Subsequent analytic techniques include, but are not limited to genomics, proteomics and lipidomics. Genomic analysis includes, but is not limited to, gene profiling and identification, analysis of methylation patterns, mutation analysis, polymorphism analysis and gene expression analysis. Proteomic analysis includes, but is not limited to, polypeptide profiling and identification, mutation analysis, polypeptide modification analysis and polypeptide expression analysis. Lipidomic analysis includes, but is not limited to, lipid profiling and identification, lipid modification analysis and lipid expression analysis.

Example 3—Characterization of Isolated Fractions

FIG. 1B shows a representative chromatogram and quantitative peak analysis of the nucleic acid isolated by the methods described herein. FIG. 1B shows 2 separate samples, designated VAP3 (leftmost graph) and VAP4 (rightmost graph). As can be seen in FIG. 1B, the samples generate a major peak and a minor peak; the major peak corresponds to fraction 13 collected from the density gradient as described below and contains all or majority of the circulating cell-free nucleic acid and cell free complex. Table 1 shows the quantitative measurements for each histogram. As shown in Table 1, the various characteristics of the histograms can be quantitated using the variables described; other variables may also be used as is known in the art. From these variables, an index factor can be generated for each sample to characterize each sample and allowing the ultimate differentiation of healthy subjects and subjects that may be suffering from or pre-disposed to a particular disease or condition. Such an index factor may be generated by using the variables shown in Table 1 or other variables known in the art. For example, the index factor may be calculated by subtracting the height of the major and minor peaks and dividing this difference by the difference of the retention time of the major and minor peaks. Using the values in Table 1 for the VAP3 sample, the calculation would be (372.65−75.01)/(18.03−20.41). Alternatively, another variable, such as the width of the major and minor peaks may be used. Using the values in Table 1 for the VAP3 sample, the calculation would be (257.6−122.6)/(18.03−20.41). The above are exemplary only and other variables or combinations of variables may be used to characterize the samples. In addition, specific properties of the circulating cell-free nucleic acid and/or the cell free complex or the components thereof, such as the presence of one or more mutations, polymorphisms, post-translational modifications and the like may also be used, either alone or in combination with the characteristics described above, to characterize a sample.

For FIG. 1B, samples were prepared as described in Example 1. Conditions for density gradient preparation and centrifugation conditions were: CsCl density gradient providing a final density of 1.32 to 1.40 g/cm³; 3 hour spin at 65,0000 rpm at 20° C. using a Beckman Coulter Optima XL-100K ultracentrifuge and Beckman VTI 65.2 rotor (416,000×g). Fractions (250 ul) were collected from the bottom of the centrifuge vessel using a peristaltic pump; 20 fractions were collected. Under these conditions, fraction 13 contains the circulating cell-free nucleic acid/cell free complex comprising circulating, cell free nucleic acid bound to cellular components.

Ret

High

Width

Asym
Trailing

Sample
Time
Area
Area %
Height
Value
Width
50%
Res
10%
10%

VAP3
18.03
35525.77
14.93
372.65
367.78
257.6
80.8
0.876
1.6
1.3

20.41
3188.21
1.34
75.01
70.28
122.6
245.2
0.744
0.1
0.55

VAP4
26.67
40841.19
17.16
362.62
357.85
275
129.4
0.507
0.65
0.82

27.94
6616.56
2.78
204.92
200.09
165.2
164.8
2.827
0.12
0.56

As discussed herein, the methods described separate circulating cell-free nucleic acid/cell free complex comprising a pool of circulating cell free nucleic acid that are associated with cellular components, such as polypeptides and lipids, from circulating cell free nucleic acid that is not associated with such cellular components (or not associated with such cellular components to an appreciable degree) and polypeptides. Each of these classes migrates in a distinct pattern during density gradient centrifugation. As described herein, the circulating cell free nucleic acid that is associated with a cellular component represents a biologically relevant fraction of such nucleic acids and as a result, the methods described herein provide a mechanism to isolate this biologically relevant pool of circulating, cell free nucleic acid for analysis.

In order to further characterize the material isolated, the plasma samples were prepared as described above and treated with various agents before being subject to density gradient centrifugation (conditions for sample preparation, density gradient preparation and centrifugation were as described for FIG. 1B). Chromatograms were generated by monitoring ultraviolet absorbance at 260 nm (detecting nucleic acids and polypeptides). Samples were treated with DNase I (1-1000 μg/ml for 1 hr at 37° C.) to non-specifically cleave double stranded DNA, DNase (1-1000 μg/ml for 1 hr at 37° C.) and RNase A (1-1000 μg/ml for 1 hr at 37° C.) to non-specifically cleave RNA and proteinase K (50-250 μg/ml for 1-4 hrs at 56° C.) to non-specifically cleave polypeptides. Reactions with DNase, RNase and proteinase K were carried out according to manufacturers' instructions. As shown in FIG. 2, the leftmost chromatogram illustrates no treatment, followed by DNase I treatment, DNase I and RNase A treatment, and proteinase K treatment (rightmost chromatogram). As can be seen treatment with each agent modified the resulting chromatogram indicating that each of DNA, RNA and nucleic acid/polypeptide complexes polypeptides are present in the material isolated by the methods described herein.

In FIG. 3, the conditions for sample preparation, density gradient preparation and centrifugation were as described for FIG. 1B. In this FIG. the peak fraction for each sample (fraction 13) were collected and desalted to remove CsCl salt using a Microcon DNA Fast Flow Centrifugal Filter Unit and re-suspended in 25 μl of water. Samples re-suspended in water were treated with DNase I (1-1000 μg/ml for 1 hr at 37° C.) to non-specifically cleave double stranded DNA, DNase I (1-1000 μg/ml for 1 hr at 37° C.) and RNase A (1-1000 μg/ml for 1 hr at 37° C.) to non-specifically cleave both DNA and RNA, mung bean nuclease (1-5 U/ug DNA) to cleave single stranded nucleic acid and proteinase K (50-250 μg/ml for 1-4 hrs at 56° C.) to non-specifically cleave polypeptides. The treated samples were subject to agarose gel electrophoresis according to standard procedures. Reactions with DNase I, RNase A, mung bean nuclease and proteinase K were carried out according to manufacturers' instructions. The gel in FIG. 3 shows molecular weight markers in lane 1, control (no treatment) sample in lane 2, sample treated with DNase I in lane 3, sample treated with DNase I and RNase A in lane 4, sample treated with mung bean nuclease in lane 5 and sample treated with proteinase K in lane 6. Agarose gel was run under standard conditions and stained with ethidium bromide. As shown in FIG. 3, treatment with each agent modified the resulting chromatogram indicating that each of double and single stranded DNA, RNA and nucleic acid/polypeptide complexes are present in the material isolated by the methods described herein.

Example 4—Comparison of the Methods of the Present Disclosure with Commercially Available Extraction Kits

Table 2 shows a comparison of the methods of the present disclosure with two commercially available kits for the isolation of circulating cell free nucleic acid. Procedures for the commercially available kits were carried out according to manufacturers' instructions. The commercially available kits were the NucleoSpin Kit from Clontech and the QIAamp Circulating Nucleic Acid Kit from Qiagen.

The QIAamp Circulating Nucleic Acid Kit simplifies isolation of circulating DNA and RNA from plasma or serum. No phenol-chloroform extraction is required and nucleic acids bind specifically to the QIAamp Mini column, while contaminants pass through. PCR inhibitors, such as divalent cations and proteins, are completely removed in 3 wash steps, leaving pure nucleic acids to be eluted in a buffer provided with the kit. The QIAamp Circulating Nucleic Acid technology yields circulating DNA and RNA from human plasma, serum, or urine. Circulating RNA can be purified with DNA digestion using the RNase-Free DNase Set.

The NucleoSpin Plasma XS kit is designed to isolate fragmented DNA larger than 50 bp from human EDTA blood plasma. NucleoSpin Plasma XS allows elution in only 5-20 μl, resulting in highly concentrated DNA.

For the methods of the present disclosure, sample preparation, density gradient preparation and centrifugation were carried out as described for FIG. 1B above. As can be seen, the methods of the present disclosure provide a method that is easier to implement, requires less sample volume and provides a significant increase in yield and purity. Furthermore, the methods provided herein offer a significant throughput for sample processing.

TABLE 2

QIAamp

Circulating

NucleoSpin Kit
Nucleic
Present

(Clontech)
Acid Kit (Qiagen)
Disclosure

Throughput
30 min/6
2 hr/24
3 hr/16

samples
samples
samples

Sample Volume
200 μl
200 μl
20 μl

RNA Carrier
No
Yes
No

Required

Large Volume to
Yes
Yes (up to 5 ml)
No

Improve Yield

Yield, range
0.61 to 50.11
9.43 to 46.27
97.30 to 132.25

(ng/μl) n = 6

Yield, means ±
12.58 ± 18.68
18.58 ± 13.80
108.46 ± 11.17

SD, n = 6

Yield, % CV,
148.42
74.26
10.30

n = 6

Purity (A260/
>1.8
>2.0
0.57 to 0.63

280), n = 6

FIG. 4 shows representative agarose gels of circulating cell free DNA isolated using the methods of the present disclosure (as described in FIG. 1B) (designated V) and that prepared using the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). Fraction 13 is shown. Agarose gel was run under standard conditions and stained with ethidium bromide. In FIG. 4, three separate samples are show, with lanes M and 10 being molecular weight markers, lanes 1, 4 and 7 corresponding to DNA isolated using the NucleoSpin kit from samples 1-3, respectively, lanes 2, 5, and 8 corresponding to DNA isolated using the QIAamp kit from samples 1-3, respectively and lanes 3, 6 and 9 corresponding to DNA isolated using the methods of the present disclosure from samples 1-3, respectively. The difference in yield of DNA isolated is evident when comparing lanes 3, 6 and 9 to the remaining lanes.

Example 5—Detection of Gene Polymorphisms from Circulating, Cell Free DNA

This example illustrates the detection of multiple gene polymorphisms on circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available kits of Example 4. For the methods of the present disclosure, circulating cell free DNA was prepared as described in for FIG. 1B, with the addition of a desalting step and disassociation of nucleoprotein complexes as described in Example 1. The commercially available kits were used according to manufacturers' instructions. Isolated DNA was used directly for genotyping experiments.

This example shows the detection of 7 single nucleotide polymorphisms (SNP) over a single gene for PCSK9, 3 SNPs over 2 genes related to warfarin sensitivity, 11 SNP's in a single gene for plavix sensitivity and 4 SNPs over 3 genes related to thrombophilia risk. The PCSK9 genotyping test was developed by the Applicants; genotyping for warfarin sensitivity, plavix sensitivity and thrombophilia risk were carried out using commercially available kits from GenmarkDx using the eSensor platform.

Table 3 shows the general PCR conditions used in the genotyping experiments while Table 4 shows the probes used in the PCSK9 genotyping experiments. Probes and primers for the commercially available kits are not available to the general public.

TABLE 3

Reference

SNP

Gene Name
SNP ID
Allele
PCR Conditions

PCSK9

PCSK9
rs562556
A > G
95° C.^{10 min}× 1 cycle;

PCSK9
rs505151
A > G
(92° C.^{15 sec}-60° C.^{1 min}) ×

PCSK9
rs11206510
T > C
50 cycles

PCSK9
rs1151147
G > T

PCSK9
rs28362286
C > A

PCSK9
rs28362263
G > A

PCSK9
rs7517090
G > A

Thrombophilia Risk

Factor II
20210
G > A
95° C.^{4 min}× 1 cycle;

(Prothrombin)

(95° C.^{25 sec}-60° C.^{30 sec}-

Factor V
1691
G > A
72° C.^{25 sec}) ×

(Leiden)

35 cycles

MTHFR
677
C > T

1298
A > C

Warfarin Sensitivity

CYP450 2C19
430
C > T
95° C.^{4 min}× 1 cycle;

*2, *3
1075
A > C
(93° C.^{45 sec}-56° C.^{45 sec}-

VKORC1
−1639
G > A
68° C.^{45 sec}) ×

39 cycles;

68° C.^{7 min}× 1 cycle

Plavix Sensitivity

CYP450 2C19
681
G > A
94° C.^{4 min}× 1 cycle;

CYP450 2C19
636
G > A
(94° C.^{20 sec}-56° C.^{45 sec}-

CYP450 2C19
1
A > G
70° C.^{45 sec}) ×

CYP450 2C19
1297
C > T
37 cycles;

CYP450 2C19
396
G > A

CYP450 2C19
19249
T > A

CYP450 2C19
358
T.C

CYP450 2C19
12784
G > A

CYP450 2C19
19153
C > T

CYP450 2C19
87290
C > T

CYP450 2C19
−806
C > T

TABLE 4

Gene Name
SNP ID
Taqman Probe

PCSK9
rs562556
GGGGCCTACACGGATGGCCACAGCC[A/G]TCG

CCCGCTGCGCCCCAGATGAGGA

PCSK9
rs505151
AGCACTACAGGCAGCACCAGCGAAG[A/G]GG

CCGTGACAGCCGTTGCCATCTGC

PCSK9
rs11206510
AAGGATATAGGGAAAACCTTGAAAG[C/T]GA

TGTCTGTGGTGGCCGTCTTTGGC

PCSK9
rs11591147
TACGAGGAGCTGGTGCTAGCCTTGC[G/T]TTC

CGAGGAGGACGGCCTGGCCGAA

PCSK9
rs28362286
CCGTGACAGCCGTTGCCATCTGCTG[A/C]CGG

AGCCGGCACCTGGCGCAGGCCT

PCSK9
rs28362263
GGTACTGACCCCCAACCTGGTGGCC[A/G]CCC

TGCCCCCCAGCACCCATGGGGC

PCSK9
rs7517090
GAGTGTGGCCTGTGCAGAAGGGACC[A/G]AG

GCTGGTGAGACCAGGAGGGCCTG

Table 5 shows the results of the genotyping experiments using the circulating, cell free DNA isolated by the methods of the present disclosure (designated V) and the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). The data in Table 5 shows that genotyping experiments using circulating cell free DNA purified by the methods of the present disclosure allowed the accurate determination of SNP genotype for all 25 SNPs tested.

TABLE 5

Kit C
Kit Q
V

PCSK9, n = 6
95.2%
90.5%
100%

Warfarin Sensitivity, n = 6
66.7%
100%
100%

Plavix Sensitivity, n = 6
83.3%
100%
100%

Thrombophilia Risk, n = 6
83.3%
100%
100%

Example 6—Detection of KRAS Gene Mutations from Circulating, Cell Free DNA

This example illustrates the detection of KRAS gene mutations on circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available kits of Example 4. For the methods of the present disclosure, circulating cell free DNA was prepared as described in for FIG. 1B, with the addition of a desalting step and disassociation of nucleoprotein complexes as described in Example 1. The commercially available kits were used according to manufacturers' instructions. Isolated DNA was used directly for genotyping experiments.

The KRAS mutation is found in several cancers including colorectal, lung, thyroid, and pancreatic cancers and cholangiocarcinoma. KRAS mutations are often located within codons 12 and 13 of exon 2, which may lead to abnormal growth signaling by the p21-ras protein. These alterations in cell growth and division may trigger cancer development as signaling is excessive. A KRAS mutation often serves as a useful prognostic marker of drug response. For example, a KRAS mutation is considered to be a strong prognostic marker of response to tyrosine kinase inhibitors such as gefitinib (Iressa) or erlotinib (Tarceva). Recently, KRAS mutations have been detected in many colorectal cancer patients and may be associated with responses to cetuximab (Erbitux) or panitumumab (Vectibix), which are used in colon cancer therapy Mutations in KRAS codons 12 and 13 have been associated with lack of response to EGFR-targeted therapies in both colorectal cancer and non-small cell lung cancer patients. The National Comprehensive Cancer Network recommends KRAS mutation testing before initiating EGFR-targeted therapies for such diseases.

KRAS mutation analysis was performed using (PNAClamp KRAS Mutation Detection Kit; Panagene, Daejeon, Korea). The mutation analysis was performed according to manufactures' instructions. Table 6 shows the results of the KRAS mutation experiments using the circulating, cell free DNA isolated by the methods of the present disclosure (designated V) and the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). PCR conditions were: 94° C.^{5 min}×1 cycle; (94° C.^30sec−70° C.^20sec−63° C.^30sec−72° C.^30sec)×40 cycles.

The data in Table 6 shows that KRAS mutation analysis using circulating cell free DNA purified by the methods of the present disclosure allowed the accurate determination of KRAS mutation status that was superior to the results obtained with commercially available kit Q.

TABLE 6

Kit C
Kit Q
V

KRAS Mutation
ND
4/14 detected
9/14 detected

Status (Codon

12/13); n = 7

Example 7—Detection of TP53 Exon Amplification Products from Circulating, Cell Free DNA

This example illustrates the detection of TP53 exon amplification products from circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available kits of Example 4. For the methods of the present disclosure, circulating cell free DNA was prepared as described in for FIG. 1B, with the addition of a desalting step and disassociation of nucleoprotein complexes as described in Example 1. The commercially available kits were used according to manufacturers' instructions. Isolated DNA was used directly for genotyping experiments. The TP53 gene provides instructions for making tumor protein p53. This protein acts as a tumor suppressor, which regulates cell division by keeping cells from growing and dividing too fast or in an uncontrolled way. Identifying mutations in the TP53 gene is important for diagnostic, prognostic and monitoring purposes and can guide in the selection of appropriate therapeutic intervention. TP53 mutations are found associated with drug resistance to platinum-based chemotherapy without any effect on the sensitivity of paclitaxel. Thus, detection and analysis of gene mutations of TP53 have become a guide for cancer individualized chemotherapy.

TP53 exon amplification analysis was performed on exon 1 of TP53 using (Multiplex PCR kit for Human TP53 Oncogene from Bio SB). The analysis was performed according to manufactures' instructions. PCR conditions were: 95° C.^{5 min}×1 cycle; (95° C.^25sec−64° C.^20sec−72° C.^20sec)×15 cycles; (93° C.^25sec−60° C.^35sec−72° C.^20sec)×28 cycles.

Table 7 shows the results of the TP53 exon amplification experiments using the circulating, cell free DNA isolated by the methods of the present disclosure (designated V) and the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). The data in Table 7 shows that exon amplification of the TP53 gene using circulating cell free DNA purified by the methods of the present disclosure was equal to (Kit Q) or superior to (Kit C) the results obtained with commercially available kit Q.

TABLE 7

Kit C
Kit Q
V

TP53 Exon
+
+++
+++

Amplification (392-

1,143 bp), n = 6

Example 8—Detection of Short Tandem Repeat DNA Profiling from Circulating, Cell Free DNA

This example illustrates the detection of short tandem repeat (STR) profiling from circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available kits of Example 4. STRs are highly polymorphic, repeating sequences and represent non-coding DNA sequences. STRs are frequently used in determining identity. As STRs are non-coding DNA regions, the nucleic acid containing such STRs should not be enriched in the methods of the present disclosure. For the methods of the present disclosure, circulating cell free DNA was prepared as described in for FIG. 1B, with the addition of a desalting step and disassociation of nucleoprotein complexes as described in Example 1. The commercially available kits were used according to manufacturers' instructions. Isolated DNA was used directly for genotyping experiments.

16 STRs were analyzed using the AmpFLSTR Identifiler PCR Amplification kit. This kit allows the determination of 15 tetranucleotide repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D351358, TH01, D13S317, D165539, D21S1338, D195433, vWA, TPDX, D18S51, D5S818, FGA and the Amelogenin gender-determining marker) in a single PCR amplification. Reactions were carried out according to manufacturers' instructions on circulating, cell free DNA isolated using the methods of the present disclosure and circulating, cell free DNA isolated using the commercial kits described above. PCR conditions were: 95° C.^11min×1 cycle; (94° C.^60sec−59° C.^60sec−72° C.^60sec)×28 cycles; 60° C.^60min×1 cycle.

Table 8 shows the results of the STR profiling experiments using the circulating, cell free DNA isolated by the methods of the present disclosure (designated V) and the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). The data in Table 8 shows that STR profiling using circulating cell free DNA isolated using the methods of the present disclosure was less effective, as expected, than using circulating cell free DNA isolated using Kit Q in detection of these non-coding STR markers.

TABLE 8

(;

Kit C
Kit Q
V

STR DNA Profiling;
ND
6/16, 9/15,
0/16, 8/15,

n = 4

4/16, 0/16
1/16, 0/16

Example 9—Array Comparative Genomic Hybridization from Circulating, Cell Free DNA

This example illustrates the use of array comparative genomic hybridization (aCGH) from circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available kits of Example 4. For the methods of the present disclosure, circulating cell free DNA was prepared as described in for FIG. 1B, with the addition of a desalting step and disassociation of nucleoprotein complexes as described in Example 1. The commercially available kits were used according to manufacturers' instructions. Isolated DNA was used directly for genotyping experiments.

In this example, the InfiniumDx CytoSNP-12 Assay (Illumina) was used to screen over 294,000 SNP markers over the entire genome. Reactions were carried out according to manufacturers' instructions on circulating, cell free DNA isolated using the methods of the present disclosure and circulating, cell free DNA isolated using the commercial kits described above. aCGH analysis is useful in detecting genomic structural variation such as copy number changes, copy neutral loss of heterozygosity, uniparental disomy, mitotic recombination, gene conversion and mosaicism.

Table 9 shows the results of the aCGH experiments using the circulating, cell free DNA isolated by the methods of the present disclosure (designated V) and the two commercially available kits described above (designated C for the NucleoSpin Kit and Q for the QIAamp kit). The data in Table 9 shows that aCGH SNP profiling of whole human genome using circulating cell free DNA purified by the methods of the present disclosure was more representative of cell genomic DNA than using circulating cell free DNA isolated with Kit Q.

TABLE 9

Kit C
Kit Q
V

Array GCH
ND
Poorly
Highly

Analysis; n = 4

Representative
Representative

FIG. 5 shows representative results of aCGH of individual chromosomes using circulating cell free DNA isolated using the methods of the present disclosure as well as comparing such detection using circulating cell free DNA isolated using the commercially available Kit Q of Example 4. The individual chromosomes shown are chromosome 1 (upper panel), chromosome 6 (middle panel) and chromosome 17 (lower panel). The leftmost column shows the aCGH analysis performed using circulating, cell free DNA isolated using Kit Q, the middle column shows the aCGH analysis performed using circulating, cell free DNA isolated using the methods of the present disclosure and the rightmost column shows the aCGH analysis performed using genomic DNA (reference gDNA). As can be seen, the results obtained using circulating, cell free DNA isolated by the method of the present disclosure provided superior results when compared to results obtained using circulating, cell free DNA isolated by Kit Q and were in agreement with the results obtained using genomic DNA.

FIG. 6 shows the results from all 22 autosomes and each allosome. The lower panel shows the aCGH analysis performed using circulating, cell free DNA isolated using Kit Q, the middle panel shows the aCGH analysis performed using circulating, cell free DNA isolated using the methods of the present disclosure and the upper panel shows the aCGH analysis performed using genomic DNA (reference gDNA). As can be seen, the results obtained using circulating, cell free DNA isolated by the method of the present disclosure provided superior results when compared to results obtained using circulating, cell free DNA isolated by Kit Q and were in agreement with the results obtained using genomic DNA.

The results in Table 9 and FIGS. 5 and 6 show that the nucleic acid isolated using the methods of the present disclosure is highly representative of genomic DNA.

Example 10—Analysis of Lipid and Polypeptide Composition

As discussed herein, the nucleic acids isolated according to the methods of the present disclosure are associated with cellular components, such as but not limited to, polypeptides and lipids. These associated cellular components aid in protecting the nucleic acids from degradation and provide an additional source of information regarding the subject. As a result, the biological information that can be obtained from the material isolated as described herein is greater than obtainable with commercially available kits which extract only the nucleic acids. To investigate the nature of the associated cellular components that are isolated with the circulating cell free nucleic acid, circulating cell free nucleic acid and its associated cellular components were isolated as described for FIG. 1B.

The isolated material was subject to Vertical Auto Profile (VAP) analysis as described in Example 1 to identify the nature of the lipid component associated with the nucleic acid. Nucleic acid/histone complexes were detected in density gradient fractions using the Cell Death Detection ELISA kit (Roche, catalogue number 1774425). This assay detects histone associated DNA complexes, both mono- and loig-onucleosomes, using a photometric enzyme immunoassay and was used to determine the fractions containing DNA associated with cellular components. As shown in FIG. 7, triglyceride was the only lipid detected in the isolated complexes, with the triglyceride being detected only in those fractions where polypeptide associated DNA was detected. The solid line in FIG. 7 indicates absorbance at 260 nm.

In addition, polypeptide components associated with the isolated nucleic acid were analyzed by LC-MS/MS analysis to determine the identity of the associated polypeptides. As shown in Table 10, over 700 associated polypeptides were identified, indicating that a variety of polypeptides are associated with the isolated nucleic acids.

TABLE 10

Accession
Molecular

Identified Proteins
Taxonomy
RepID
Number
Weight

14-3-3 protein epsilon
Amniota
1433E_HUMAN
P62258
29 kDa

14-3-3 protein eta
Simiiformes
1433F_HUMAN
Q04917
28 kDa

(+1)

14-3-3 protein gamma
Amniota
1433G_HUMAN
P61981
28 kDa

14-3-3 protein sigma
Hominoidea
1433S_HUMAN
P31947
28 kDa

14-3-3 protein theta
Eutheria
1433T_HUMAN
P27348
28 kDa

14-3-3 protein zeta/delta
Amniota
1433Z_HUMAN
P63104
28 kDa

26S protease regulatory

Homo sapiens

E9PM69_HUMAN
E9PM69
44 kDa

subunit 6A

(+1)

26S protease regulatory
Eutheria
PRS7_HUMAN
P35998
49 kDa

subunit 7

26S protease regulatory
Eutheria
A8K3Z3_HUMAN
A8K3Z3
45 kDa

subunit 8

(+1)

26S proteasome non-ATPase

Homo sapiens

PSMD1_HUMAN
Q99460
106 kDa

regulatory subunit 1

26S proteasome non-ATPase
Catarrhini
PSD12_HUMAN
O00232
53 kDa

regulatory subunit 12

26S proteasome non-ATPase

Homo sapiens

B4DJ66_HUMAN
B4DJ66
35 kDa

regulatory subunit 13

(+2)

26S proteasome non-ATPase
Homininae
PSMD2_HUMAN
Q13200
100 kDa

regulatory subunit 2

(+4)

26S proteasome non-ATPase
Homininae
C9IZE4_HUMAN
C9IZE4
52 kDa

regulatory subunit 6

(+1)

28S ribosomal protein S9,

Homo sapiens

RT09_HUMAN
P82933
46 kDa

mitochondrial

3-hydroxyacyl-CoA
Homininae
HCD2_HUMAN
Q99714
27 kDa

dehydrogenase type-2

3-ketoacyl-CoA thiolase
Catarrhini
B4E2W0_HUMAN
B4E2W0
49 kDa

(+1)

40S ribosomal protein S11
Amniota
RS11_HUMAN
P62280
18 kDa

40S ribosomal protein S12
Mammalia
RS12_HUMAN
P25398
15 kDa

40S ribosomal protein S14
Euteleostomi
RS14_HUMAN
P62263
16 kDa

40S ribosomal protein S15a
Mammalia
RS15A_HUMAN
P62244
15 kDa

40S ribosomal protein S18
Euarchontoglires
Q5GGW2_HUMAN
Q5GGW2
4 kDa

40S ribosomal protein S2

Homo sapiens

E9PQD7_HUMAN
E9PQD7
25 kDa

(+2)

40S ribosomal protein S20

Homo sapiens

E5RIP1_HUMAN
E5RIP1
5 kDa

(+2)

40S ribosomal protein S24

Homo sapiens

E7EPK6_HUMAN
E7EPK6
32 kDa

(+1)

40S ribosomal protein S25
Amniota
RS25_HUMAN
P62851
14 kDa

40S ribosomal protein S3
Eukaryota
RS3_HUMAN
P23396
27 kDa

40S ribosomal protein S3a
Eutheria
RS3A_HUMAN
P61247
30 kDa

40S ribosomal protein S4, X
Eutheria
RS4X_HUMAN
P62701
30 kDa

isoform

40S ribosomal protein S5
Mammalia
RS5_HUMAN
P46782
23 kDa

40S ribosomal protein S7
Theria
RS7_HUMAN
P62081
22 kDa

40S ribosomal protein S9
Eutheria
RS9_HUMAN
P46781
23 kDa

4F2 cell-surface antigen

Homo sapiens

B4E2Z3_HUMAN
B4E2Z3
56 kDa

heavy chain

(+2)

5-aminoimidazole-4-

Homo sapiens

A8K202_HUMAN
A8K202
65 kDa

carboxamide ribonucleotide

(+1)

formyltransferase/IMP

cyclohydrolase, isoform

CRA_g

5-azacytidine induced 1

Homo sapiens

B2RN10_HUMAN
B2RN10
122 kDa

(+1)

60 kDa heat shock protein,

Homo sapiens

CH60_HUMAN
P10809
61 kDa

mitochondrial

60S acidic ribosomal protein

Homo sapiens

F8VWS0_HUMAN
F8VWS0
30 kDa

P0

(+2)

60S acidic ribosomal protein
Catarrhini
RLA2_HUMAN
P05387
12 kDa

P2

60S ribosomal protein L10
Euarchontoglires
F8W7C6_HUMAN
F8W7C6
19 kDa

(+1)

60S ribosomal protein L10a
Eutheria
RL10A_HUMAN
P62906
25 kDa

60S ribosomal protein L12
Eutheria
RL12_HUMAN
P30050
18 kDa

60S ribosomal protein L13

Homo sapiens

RL13_HUMAN
P26373
24 kDa

60S ribosomal protein L13a
Hominidae
RL13A_HUMAN
P40429
24 kDa

(+1)

60S ribosomal protein L17
Homininae
B4E3C2_HUMAN
B4E3C2
17 kDa

(+1)

60S ribosomal protein L18

Homo sapiens

B4DDY5_HUMAN
B4DDY5
16 kDa

(+3)

60S ribosomal protein L18a

Homo sapiens

B2R4C0_HUMAN
B2R4C0
21 kDa

(+2)

60S ribosomal protein L19
Theria
RL19_HUMAN
P84098
23 kDa

60S ribosomal protein L23
Euteleostomi
RL23_HUMAN
P62829
15 kDa

60S ribosomal protein L23a
Eutheria
RL23A_HUMAN
P62750
18 kDa

60S ribosomal protein L24
Eutheria
RL24_HUMAN
P83731
18 kDa

60S ribosomal protein L28
Catarrhini
RL28_HUMAN
P46779
16 kDa

60S ribosomal protein L3
Catarrhini
RL3_HUMAN
P39023
46 kDa

60S ribosomal protein L32
Homininae
F8W727_HUMAN
F8W727
18 kDa

(+1)

60S ribosomal protein L4

Homo sapiens

E7EWF1_HUMAN
E7EWF1
46 kDa

(+1)

60S ribosomal protein L5
Hominoidea
RL5_HUMAN
P46777
34 kDa

60S ribosomal protein L6
Homininae
RL6_HUMAN
Q02878
33 kDa

60S ribosomal protein L7
Homininae
RL7_HUMAN
P18124
29 kDa

60S ribosomal protein L7a
Eutheria
RL7A_HUMAN
P62424
30 kDa

60S ribosomal protein L8

Homo sapiens

E9PIZ3_HUMAN
E9PIZ3
24 kDa

(+1)

6-phosphofructokinase

Homo sapiens

Q6ZTT1_HUMAN
Q6ZTT1
93 kDa

(+1)

6-phosphofructokinase type

Homo sapiens

K6PP_HUMAN
Q01813
86 kDa

C

6-phosphofructokinase, liver

Homo sapiens

K6PL_HUMAN
P17858
85 kDa

type

6-phosphogluconate

Homo sapiens

6PGD_HUMAN
P52209
53 kDa

dehydrogenase,

(+1)

decarboxylating

78 kDa glucose-regulated
Catarrhini
GRP78_HUMAN
P11021
72 kDa

protein

7-dehydrocholesterol

Homo sapiens

DHCR7_HUMAN
Q9UBM7
54 kDa

reductase

ABC50 protein

Homo sapiens

Q2L6I2_HUMAN
Q2L6I2
92 kDa

(+1)

Abhydrolase domain-

Homo sapiens

B4DNR3_HUMAN
B4DNR3
20 kDa

containing protein 14B

(+2)

Acetolactate synthase-like

Homo sapiens

ILVBL_HUMAN
A1L0T0
68 kDa

protein

Acetyl-CoA
Homininae
THIL_HUMAN
P24752
45 kDa

acetyltransferase,

mitochondrial

Aconitase 2, mitochondrial

Homo sapiens

A2A274_HUMAN
A2A274
88 kDa

(+2)

Actin, cytoplasmic 1, N-
Simiiformes
B4E335_HUMAN
B4E335
39 kDa

terminally processed

(+3)

Actin-related protein 2/3

Homo sapiens

C9JWM7_HUMAN
C9JWM7
22 kDa

complex subunit 4

(+3)

ADAM metallopeptidase
Catarrhini
A8MY20_HUMAN
A8MY20
63 kDa

domain 10, isoform CRA_a

(+1)

Adaptor-related protein

Homo sapiens

H0UID3_HUMAN
H0UID3
105 kDa

complex 2, beta 1 subunit,

(+3)

isoform CRA_d

Adenine

Homo sapiens

H3BQZ9_HUMAN
H3BQZ9
17 kDa

phosphoribosyltransferase

(+1)

Adenosylhomocysteinase
Homininae
SAHH_HUMAN
P23526
48 kDa

(+1)

Adenylosuccinate synthetase

Homo sapiens

B4E1L0_HUMAN
B4E1L0
48 kDa

(+1)

Adenylyl cyclase-associated

Homo sapiens

B2RDY9_HUMAN
B2RDY9
52 kDa

protein

(+1)

ADP/ATP translocase 2

Homo sapiens

ADT2_HUMAN
P05141
33 kDa

(+1)

ADP-ribosylation factor 3
Euteleostomi
ARF3_HUMAN
P61204
21 kDa

(+2)

ADP-ribosylation factor 6

Homo sapiens

Q5U025_HUMAN
Q5U025
20 kDa

Afamin

Homo sapiens

AFAM_HUMAN
P43652
69 kDa

A-kinase anchor protein 13

Homo sapiens

H0Y4V5_HUMAN
H0Y4V5
305 kDa

(+3)

Alanine--tRNA ligase,

Homo sapiens

SYAC_HUMAN
P49588
107 kDa

cytoplasmic

Alcohol dehydrogenase class-

Homo sapiens

ADHX_HUMAN
P11766
40 kDa

3

(+2)

Aldehyde dehydrogenase X,

Homo sapiens

AL1B1_HUMAN
P30837
57 kDa

mitochondrial

Aldose reductase

Homo sapiens

ALDR_HUMAN
P15121
36 kDa

Alpha-1-antichymotrypsin

Homo sapiens

AACT_HUMAN
P01011
48 kDa

Alpha-1-antitrypsin

Homo sapiens

A1AT_HUMAN
P01009
47 kDa

Alpha-1B-glycoprotein

Homo sapiens

A1BG_HUMAN
P04217
54 kDa

Alpha-2-antiplasmin
Homininae
A2AP_HUMAN
P08697
55 kDa

Alpha-2-macroglobulin

Homo sapiens

A2MG_HUMAN
P01023
163 kDa

Alpha-actinin-1

Homo sapiens

ACTN1_HUMAN
P12814
103 kDa

Alpha-actinin-4
Catarrhini
ACTN4_HUMAN
O43707
105 kDa

Alpha-aminoadipic

Homo sapiens

E7EPT3_HUMAN
E7EPT3
54 kDa

semialdehyde dehydrogenase

(+1)

Alpha-enolase
Catarrhini
ENOA_HUMAN
P06733
47 kDa

Aminoacyl tRNA synthase

Homo sapiens

AIMP1_HUMAN
Q12904
34 kDa

complex-interacting

multifunctional protein 1

Aminoacyl tRNA synthase

Homo sapiens

F8W950_HUMAN
F8W950
28 kDa

complex-interacting

(+2)

multifunctional protein 2

Aminopeptidase B

Homo sapiens

AMPB_HUMAN
Q9H4A4
73 kDa

Angiotensinogen

Homo sapiens

ANGT_HUMAN
P01019
53 kDa

(+2)

Ankyrin repeat domain-

Homo sapiens

ANR31_HUMAN
Q8N7Z5
211 kDa

containing protein 31

Ankyrin-3

Homo sapiens

ANK3_HUMAN
Q12955
480 kDa

Annexin

Homo sapiens

B7Z8A7_HUMAN
B7Z8A7
72 kDa

(+1)

Annexin

Homo sapiens

B2R657_HUMAN
B2R657
53 kDa

Annexin A1
Homininae
ANXA1_HUMAN
P04083
39 kDa

Annexin A2
Catarrhini
ANXA2_HUMAN
P07355
39 kDa

Annexin A3

Homo sapiens

ANXA3_HUMAN
P12429
36 kDa

Annexin A4

Homo sapiens

ANXA4_HUMAN
P09525
36 kDa

Annexin A5
Homininae
ANXA5_HUMAN
P08758
36 kDa

Antithrombin-III

Homo sapiens

ANT3_HUMAN
P01008
53 kDa

AP-3 complex subunit beta-1

Homo sapiens

AP3B1_HUMAN
O00203
121 kDa

AP-3 complex subunit mu-1
Eutheria
AP3M1_HUMAN
Q9Y2T2
47 kDa

Apolipoprotein A-I
Homininae
APOA1_HUMAN
P02647
31 kDa

Apolipoprotein A-II
Homininae
APOA2_HUMAN
P02652
11 kDa

Apolipoprotein A-IV

Homo sapiens

APOA4_HUMAN
P06727
45 kDa

Apolipoprotein B (Including

Homo sapiens

C0JYY2_HUMAN
C0JYY2
516 kDa

Ag(X) antigen)

Apolipoprotein C-III

Homo sapiens

APOC3_HUMAN
P02656
11 kDa

Apolipoprotein D

Homo sapiens

APOD_HUMAN
P05090
21 kDa

Apolipoprotein E

Homo sapiens

APOE_HUMAN
P02649
36 kDa

Apolipoprotein F

Homo sapiens

APOF_HUMAN
Q13790
35 kDa

Apolipoprotein L1

Homo sapiens

E9PF24_HUMAN
E9PF24
42 kDa

(+1)

Apolipoprotein M

Homo sapiens

APOM_HUMAN
O95445
21 kDa

(+2)

Apolipoprotein(a)
Catarrhini
APOA_HUMAN
P08519
501 kDa

(+1)

Arginine--tRNA ligase,

Homo sapiens

SYRC_HUMAN
P54136
75 kDa

cytoplasmic

Asparagine synthetase

Homo sapiens

ASNS_HUMAN
P08243
64 kDa

[glutamine-hydrolyzing]

(+1)

Asparagine--tRNA ligase,

Homo sapiens

SYNC_HUMAN
O43776
63 kDa

cytoplasmic

Aspartate aminotransferase,

Homo sapiens

AATM_HUMAN
P00505
48 kDa

mitochondrial

(+1)

Aspartate--tRNA ligase,

Homo sapiens

SYDC_HUMAN
P14868
57 kDa

cytoplasmic

Aspartyl/asparaginyl beta-

Homo sapiens

ASPH_HUMAN
Q12797
86 kDa

hydroxylase

Atlastin-3
Homininae
F5H6I7_HUMAN
F5H6I7
59 kDa

(+1)

ATP synthase subunit alpha

Homo sapiens

B4DY56_HUMAN
B4DY56
58 kDa

(+1)

ATP synthase subunit beta,
Eutheria
ATPB_HUMAN
P06576
57 kDa

mitochondrial

ATP-binding cassette sub-
Homininae
E7EUE1_HUMAN
E7EUE1
78 kDa

family D member 3

(+1)

ATP-binding cassette sub-
Euarchontoglires
ABCE1_HUMAN
P61221
67 kDa

family E member 1

ATP-citrate synthase

Homo sapiens

ACLY_HUMAN
P53396
121 kDa

ATP-dependent RNA
Homininae
DHX9_HUMAN
Q08211
141 kDa

helicase A

ATP-dependent RNA

Homo sapiens

DDX25_HUMAN
Q9UHL0
55 kDa

helicase DDX25

(+1)

ATP-dependent RNA

Homo sapiens

B5BTY4_HUMAN
B5BTY4
73 kDa

helicase DDX3X

(+1)

ATP-dependent RNA

Homo sapiens

DHX29_HUMAN
Q7Z478
155 kDa

helicase DHX29

AT-rich interactive domain-

Homo sapiens

ARI4A_HUMAN
P29374
143 kDa

containing protein 4A

Basic leucine zipper and W2
Eutheria
BZW1_HUMAN
Q7L1Q6
48 kDa

domain-containing protein 1

Basigin

Homo sapiens

BASI_HUMAN
P35613
42 kDa

(+1)

Beta-2-glycoprotein 1

Homo sapiens

APOH_HUMAN
P02749
38 kDa

Beta-lactamase-like protein 2

Homo sapiens

LACB2_HUMAN
Q53H82
33 kDa

Bifunctional

Homo sapiens

SYEP_HUMAN
P07814
171 kDa

glutamate/proline--tRNA

ligase

Brain acid soluble protein 1

Homo sapiens

BASP1_HUMAN
P80723
23 kDa

BRCA1-A complex subunit
Simiiformes
BRE_HUMAN
Q9NXR7
44 kDa

BRE

Brefeldin A-inhibited

Homo sapiens

BIG1_HUMAN
Q9Y6D6
209 kDa

guanine nucleotide-exchange

protein 1

C-1-tetrahydrofolate

Homo sapiens

C1TC_HUMAN
P11586
102 kDa

synthase, cytoplasmic

C4b-binding protein alpha

Homo sapiens

C4BPA_HUMAN
P04003
67 kDa

chain

CAD protein

Homo sapiens

PYR1_HUMAN
P27708
243 kDa

Calnexin

Homo sapiens

CALX_HUMAN
P27824
68 kDa

Calpain small subunit 1
Hominidae
CPNS1_HUMAN
P04632
28 kDa

Calpain-1 catalytic subunit

Homo sapiens

CAN1_HUMAN
P07384
82 kDa

Calpain-2 catalytic subunit

Homo sapiens

H0Y323_HUMAN
H0Y323
83 kDa

(+1)

Calponin-2

Homo sapiens

B4DDF4_HUMAN
B4DDF4
33 kDa

(+3)

Calponin-3

Homo sapiens

F8WA86_HUMAN
F8WA86
31 kDa

(+1)

cAMP-dependent protein
Hominoidea
KAP0_HUMAN
P10644
43 kDa

kinase type I-alpha regulatory

subunit

Caprin-1
Catarrhini
CAPR1_HUMAN
Q14444
78 kDa

Carbonyl reductase
Homininae
CBR1_HUMAN
P16152
30 kDa

[NADPH] 1

Carboxypeptidase N subunit 2

Homo sapiens

CPN2_HUMAN
P22792
61 kDa

Casein kinase II subunit
Eutheria
CSK21_HUMAN
P68400
45 kDa

alpha

(+3)

Caspase recruitment domain-

Homo sapiens

CAR11_HUMAN
Q9BXL7
133 kDa

containing protein 11

Catenin alpha-1

Homo sapiens

F6XBD8_HUMAN
F6XBD8
103 kDa

(+1)

Catenin beta-1

Homo sapiens

B5BU28_HUMAN
B5BU28
86 kDa

(+1)

Cathepsin D

Homo sapiens

CATD_HUMAN
P07339
45 kDa

CCT7 protein

Homo sapiens

Q6IBT3_HUMAN
Q6IBT3
59 kDa

(+1)

cDNA PSEC0119 fis, clone

Homo sapiens

Q8NBL9_HUMAN
Q8NBL9
62 kDa

PLACE1002376, highly

(+1)

similar to GPI transamidase

component PIG-S

Cell division control protein
Theria
CDC42_HUMAN
P60953
21 kDa

42 homolog

Cell migration-inducing

Homo sapiens

A1KYQ7_HUMAN
A1KYQ7
105 kDa

protein 17

(+1)

Cellular apoptosis

Homo sapiens

A3RLL6_HUMAN
A3RLL6
104 kDa

susceptibility protein variant

(+1)

2

Centromere protein F

Homo sapiens

CENPF_HUMAN
P49454
368 kDa

Ceruloplasmin

Homo sapiens

CERU_HUMAN
P00450
122 kDa

Chaperonin containing TCP1,

Homo sapiens

G5E9B2_HUMAN
G5E9B2
59 kDa

subunit 8 (Theta), isoform

(+1)

CRA_a

Chloride intracellular channel
Homininae
CLIC1_HUMAN
O00299
27 kDa

protein 1

Chromobox protein homolog
Eutheria
CBX3_HUMAN
Q13185
21 kDa

3

Citrate synthase,

Homo sapiens

CISY_HUMAN
O75390
52 kDa

mitochondrial

(+1)

Citron

Homo sapiens

Q2M5E1_HUMAN
Q2M5E1
237 kDa

C-Jun-amino-terminal
Catarrhini
JIP4_HUMAN
O60271
146 kDa

kinase-interacting protein 4

Clathrin heavy chain 1
Eutheria
CLH1_HUMAN
Q00610
192 kDa

Clusterin

Homo sapiens

CLUS_HUMAN
P10909
52 kDa

Coactosin-like protein
Hominidae
COTL1_HUMAN
Q14019
16 kDa

Coagulation factor XIII A

Homo sapiens

F13A_HUMAN
P00488
83 kDa

chain

(+1)

Coatomer subunit alpha

Homo sapiens

COPA_HUMAN
P53621
138 kDa

Coatomer subunit beta

Homo sapiens

COPB_HUMAN
P53618
107 kDa

Coatomer subunit beta′

Homo sapiens

COPB2_HUMAN
P35606
102 kDa

Coatomer subunit gamma

Homo sapiens

B3KND4_HUMAN
B3KND4
76 kDa

(+2)

Coatomer subunit gamma-1
Homininae
COPG1_HUMAN
Q9Y678
98 kDa

Cofilin 1 (Non-muscle),

Homo sapiens

G3V1A4_HUMAN
G3V1A4
17 kDa

isoform CRA_a

(+1)

COL21A1 protein

Homo sapiens

B7ZLK3_HUMAN
B7ZLK3
99 kDa

(+2)

Complement C1q

Homo sapiens

C1QB_HUMAN
P02746
27 kDa

subcomponent subunit B

Complement C1q

Homo sapiens

C1QC_HUMAN
P02747
26 kDa

subcomponent subunit C

Complement C1s

Homo sapiens

A6NG18_HUMAN
A6NG18
76 kDa

subcomponent heavy chain

(+1)

Complement C3

Homo sapiens

CO3_HUMAN
P01024
187 kDa

Complement C5

Homo sapiens

CO5_HUMAN
P01031
188 kDa

Complement component 4A

Homo sapiens

A7E2V2_HUMAN
A7E2V2
193 kDa

(Rodgers blood group)

(+7)

Complement component C7

Homo sapiens

CO7_HUMAN
P10643
94 kDa

Complement component C8

Homo sapiens

CO8G_HUMAN
P07360
22 kDa

gamma chain

Complement component C9

Homo sapiens

CO9_HUMAN
P02748
63 kDa

Complement factor B

Homo sapiens

B4E1Z4_HUMAN
B4E1Z4
141 kDa

Complement factor H

Homo sapiens

CFAH_HUMAN
P08603
139 kDa

Complement factor I

Homo sapiens

CFAI_HUMAN
P05156
66 kDa

(+2)

Complement factor

Homo sapiens

B3KVK6_HUMAN
B3KVK6
39 kDa

properdin, isoform CRA_c

(+1)

COP9 constitutive

Homo sapiens

B3KST5_HUMAN
B3KST5
40 kDa

photomorphogenic homolog

(+2)

subunit 4 (Arabidopsis),

isoform CRA_a

Copine I

Homo sapiens

A6PVH9_HUMAN
A6PVH9
53 kDa

(+2)

Creatine kinase B-type

Homo sapiens

E7EUJ8_HUMAN
E7EUJ8
39 kDa

(+1)

Cullin-4B

Homo sapiens

CUL4B_HUMAN
Q13620
104 kDa

(+1)

Cullin-associated NEDD8-
Eutheria
CAND1_HUMAN
Q86VP6
136 kDa

dissociated protein 1

Cyclin-dependent kinase 1
Homininae
CDK1_HUMAN
P06493
34 kDa

Cyclin-dependent kinase 12
Hominidae
CDK12_HUMAN
Q9NYV4
164 kDa

Cysteine and glycine-rich

Homo sapiens

B3KVC9_HUMAN
B3KVC9
18 kDa

protein 1

(+1)

Cytochrome b-c1 complex

Homo sapiens

QCR2_HUMAN
P22695
48 kDa

subunit 2, mitochondrial

Cytochrome c
Hominoidea
CYC_HUMAN
P99999
12 kDa

Cytochrome c oxidase

Homo sapiens

A0S0W7_HUMAN
A0S0W7
26 kDa

subunit 2

(+80)

Cytoplasmic aconitate

Homo sapiens

ACOC_HUMAN
P21399
98 kDa

hydratase

Cytoplasmic dynein 1 heavy
Eutheria
DYHC1_HUMAN
Q14204
532 kDa

chain 1

Death-inducer obliterator 1

Homo sapiens

DIDO1_HUMAN
Q9BTC0
244 kDa

Delta(3,5)-Delta(2,4)-

Homo sapiens

ECH1_HUMAN
Q13011
36 kDa

dienoyl-CoA isomerase,

mitochondrial

Desmoglein-2

Homo sapiens

DSG2_HUMAN
Q14126
122 kDa

Desmoplakin

Homo sapiens

DESP_HUMAN
P15924
332 kDa

Developmentally-regulated
Eutheria
DRG1_HUMAN
Q9Y295
41 kDa

GTP-binding protein 1

Dihydrolipoyl
Homininae
DLDH_HUMAN
P09622
54 kDa

dehydrogenase,

mitochondrial

Dihydrolipoyllysine-residue

Homo sapiens

ODP2_HUMAN
P10515
69 kDa

acetyltransferase component

of pyruvate dehydrogenase

complex, mitochondrial

Dihydropyrimidinase-related
Catarrhini
DPYL2_HUMAN
Q16555
62 kDa

protein 2

Dipeptidyl peptidase 3

Homo sapiens

DPP3_HUMAN
Q9NY33
83 kDa

DNA damage-binding protein
Simiiformes
DDB1_HUMAN
Q16531
127 kDa

1

DNA replication licensing
Hominidae
MCM2_HUMAN
P49736
102 kDa

factor MCM2

DNA replication licensing
Homininae
MCM3_HUMAN
P25205
91 kDa

factor MCM3

DNA replication licensing

Homo sapiens

MCM4_HUMAN
P33991
97 kDa

factor MCM4

DNA replication licensing

Homo sapiens

MCM5_HUMAN
P33992
82 kDa

factor MCM5

DNA-(apurinic or

Homo sapiens

APEX1_HUMAN
P27695
36 kDa

apyrimidinic site) lyase

DNA-binding protein A

Homo sapiens

DBPA_HUMAN
P16989
40 kDa

(+1)

DNA-binding protein Ikaros
Catarrhini
IKZF1_HUMAN
Q13422
58 kDa

DNA-dependent protein

Homo sapiens

PRKDC_HUMAN
P78527
469 kDa

kinase catalytic subunit

DnaJ homolog subfamily A
Eutheria
DNJA1_HUMAN
P31689
45 kDa

member 1

DnaJ homolog subfamily B
Hominidae
DNJB1_HUMAN
P25685
38 kDa

member 1

(+1)

DnaJ homolog subfamily C

Homo sapiens

DJC13_HUMAN
O75165
254 kDa

member 13

Dolichyl-

Homo sapiens

RPN1_HUMAN
P04843
69 kDa

diphosphooligosaccharide--

(+1)

protein glycosyltransferase

subunit 1

Dolichyl-

Homo sapiens

RPN2_HUMAN
P04844
69 kDa

diphosphooligosaccharide--

protein glycosyltransferase

subunit 2

Dopamine beta-hydroxylase

Homo sapiens

DOPO_HUMAN
P09172
69 kDa

Dopamine receptor

Homo sapiens

Q4W4Y1_HUMAN
Q4W4Y1
96 kDa

interacting protein 4

(+2)

Double-stranded RNA-

Homo sapiens

DSRAD_HUMAN
P55265
136 kDa

specific adenosine deaminase

(+1)

DPYSL3 protein

Homo sapiens

Q6DEN2_HUMAN
Q6DEN2
74 kDa

Dual oxidase 2

Homo sapiens

DUOX2_HUMAN
Q9NRD8
175 kDa

Dynactin subunit 2

Homo sapiens

DCTN2_HUMAN
Q13561
44 kDa

Dynamin-1-like protein
Catarrhini
DNM1L_HUMAN
O00429
82 kDa

Dynein heavy chain 1,

Homo sapiens

DYH1_HUMAN
Q9P2D7
494 kDa

axonemal

Dynein heavy chain 2,

Homo sapiens

DYH2_HUMAN
Q9P225
508 kDa

axonemal

Dystonin

Homo sapiens

DYST_HUMAN
Q03001
861 kDa

E3 ubiquitm/ISG15 ligase

Homo sapiens

TRI25_HUMAN
Q14258
71 kDa

TRIM25

E3 ubiquitin-protein ligase

Homo sapiens

HUWE1_HUMAN
Q7Z6Z7
482 kDa

HUWE1

E3 ubiquitin-protein ligase

Homo sapiens

UBR4_HUMAN
Q5T4S7
574 kDa

UBR4

Early endosome antigen 1

Homo sapiens

EEA1_HUMAN
Q15075
162 kDa

EF-hand domain-containing

Homo sapiens

EFHD2_HUMAN
Q96C19
27 kDa

protein D2

Electron transfer flavoprotein

Homo sapiens

ETFA_HUMAN
P13804
35 kDa

subunit alpha, mitochondrial

Elongation factor 1-alpha 1
Eutheria
EF1A1_HUMAN
P68104
50 kDa

(+1)

Elongation factor 1-delta

Homo sapiens

EF1D_HUMAN
P29692
31 kDa

Elongation factor 1-gamma

Homo sapiens

EF1G_HUMAN
P26641
50 kDa

Elongation factor 2
Hominidae
EF2_HUMAN
P13639
95 kDa

Elongation factor Tu,
Homininae
EFTU_HUMAN
P49411
50 kDa

mitochondrial

Endoplasmic reticulum

Homo sapiens

ERAP1_HUMAN
Q9NZ08
107 kDa

aminopeptidase 1

Endoplasmin

Homo sapiens

ENPL_HUMAN
P14625
92 kDa

(+1)

Enoyl-CoA hydratase,

Homo sapiens

ECHM_HUMAN
P30084
31 kDa

mitochondrial

Ephrin type-A receptor 2

Homo sapiens

EPHA2_HUMAN
P29317
108 kDa

ERBB2IP protein

Homo sapiens

Q1RMC9_HUMAN
Q1RMC9
154 kDa

(+2)

Erlin-1
Homininae
ERLN1_HUMAN
O75477
39 kDa

Erlin-2
Hominidae
ERLN2_HUMAN
O94905
38 kDa

Eukaryotic initiation factor
Eutheria
IF4A1_HUMAN
P60842
46 kDa

4A-I

Eukaryotic translation
Catarrhini
IF2A_HUMAN
P05198
36 kDa

initiation factor 2 subunit 1

Eukaryotic translation

Homo sapiens

EIF3A_HUMAN
Q14152
167 kDa

initiation factor 3 subunit A

(+1)

Eukaryotic translation

Homo sapiens

EIF3B_HUMAN
P55884
92 kDa

initiation factor 3 subunit B

Eukaryotic translation
Eutheria
EIF3M_HUMAN
Q7L2H7
43 kDa

initiation factor 3 subunit M

Eukaryotic translation

Homo sapiens

IF4G1_HUMAN
Q04637
175 kDa

initiation factor 4 gamma 1

Eukaryotic translation
Homininae
IF4H_HUMAN
Q15056
27 kDa

initiation factor 4H

Eukaryotic translation

Homo sapiens

IF5_HUMAN
P55010
49 kDa

initiation factor 5

(+1)

Eukaryotic translation
Tetrapoda
IF5A1_HUMAN
P63241
17 kDa

initiation factor 5A-1

(+1)

Eukaryotic translation
Catarrhini
IF6_HUMAN
P56537
27 kDa

initiation factor 6

Exportin-1
Simiiformes
XPO1_HUMAN
O14980
123 kDa

Exportin-7
Homininae
XPO7_HUMAN
Q9UIA9
124 kDa

Exportin-T
Homininae
XPOT_HUMAN
O43592
110 kDa

Extended synaptotagmin-1

Homo sapiens

ESYT1_HUMAN
Q9BSJ8
123 kDa

Extended synaptotagmin-2

Homo sapiens

ESYT2_HUMAN
A0FGR8
102 kDa

F-actin-capping protein
Homininae
CAZA1_HUMAN
P52907
33 kDa

subunit alpha-1

F-actin-capping protein
Hominidae
CAPZB_HUMAN
P47756
31 kDa

subunit beta

Far upstream element-

Homo sapiens

FUBP2_HUMAN
Q92945
73 kDa

binding protein 2

Farnesyl pyrophosphate
Homininae
FPPS_HUMAN
P14324
48 kDa

synthase

FAS-associated factor 2

Homo sapiens

FAF2_HUMAN
Q96CS3
53 kDa

Fascin
Homininae
FSCN1_HUMAN
Q16658
55 kDa

Fatty acid synthase

Homo sapiens

FAS_HUMAN
P49327
273 kDa

Fermitin family homolog 3
Homininae
URP2_HUMAN
Q86UX7
76 kDa

Fibrillin-1

Homo sapiens

FBN1_HUMAN
P35555
312 kDa

Fibrinogen alpha chain

Homo sapiens

FIBA_HUMAN
P02671
95 kDa

Fibrinogen beta chain

Homo sapiens

FIBB_HUMAN
P02675
56 kDa

Fibrinogen gamma chain

Homo sapiens

FIBG_HUMAN
P02679
52 kDa

Fibronectin

Homo sapiens

FINC_HUMAN
P02751
263 kDa

Filamin B

Homo sapiens

B2ZZ83_HUMAN
B2ZZ83
282 kDa

(+1)

Filamin-A

Homo sapiens

FLNA_HUMAN
P21333
281 kDa

(+1)

Filamin-C

Homo sapiens

FLNC_HUMAN
Q14315
291 kDa

Flotillin-1

Homo sapiens

FLOT1_HUMAN
O75955
47 kDa

(+1)

Fructose-bisphosphate
Hominoidea
ALDOA_HUMAN
P04075
39 kDa

aldolase A

Fructose-bisphosphate
Homininae
ALDOC_HUMAN
P09972
39 kDa

aldolase C

Fumarate hydratase,

Homo sapiens

FUMH_HUMAN
P07954
55 kDa

mitochondrial

G patch domain-containing

Homo sapiens

GPTC8_HUMAN
Q9UKJ3
164 kDa

protein 8

Galectin-1
Homininae
LEG1_HUMAN
P09382
15 kDa

Gelsolin

Homo sapiens

GELS_HUMAN
P06396
86 kDa

Glucosamine 6-phosphate N-
Eutheria
GNA1_HUMAN
Q96EK6
21 kDa

acetyltransferase

Glucosamine--fructose-6-
Homininae
GFPT1_HUMAN
Q06210
79 kDa

phosphate aminotransferase

[isomerizing] 1

Glucose-6-phosphate 1-
Homininae
G6PD_HUMAN
P11413
59 kDa

dehydrogenase

Glucose-6-phosphate

Homo sapiens

G6PI_HUMAN
P06744
63 kDa

isomerase

Glucosidase 2 subunit beta

Homo sapiens

GLU2B_HUMAN
P14314
59 kDa

Glutamate dehydrogenase 1,

Homo sapiens

DHE3_HUMAN
P00367
61 kDa

mitochondrial

Glutamate--cysteine ligase

Homo sapiens

GSH1_HUMAN
P48506
73 kDa

catalytic subunit

Glutaminase kidney isoform,
Homininae
GLSK_HUMAN
O94925
73 kDa

mitochondrial

Glutamine--tRNA ligase

Homo sapiens

SYQ_HUMAN
P47897
88 kDa

Glutathione S-transferase

Homo sapiens

GSTO1_HUMAN
P78417
28 kDa

omega-1

Glutathione S-transferase P

Homo sapiens

GSTP1_HUMAN
P09211
23 kDa

Glyceraldehyde-3-phosphate
Homininae
G3P_HUMAN
P04406
36 kDa

dehydrogenase

Glycine--tRNA ligase

Homo sapiens

SYG_HUMAN
P41250
83 kDa

Glycogen phosphorylase,

Homo sapiens

PYGB_HUMAN
P11216
97 kDa

brain form

Glycogen phosphorylase,
Homininae
PYGL_HUMAN
P06737
97 kDa

liver form

Golgi apparatus protein 1

Homo sapiens

GSLG1_HUMAN
Q92896
135 kDa

Guanine nucleotide-binding
Tetrapoda
GBLP_HUMAN
P63244
35 kDa

protein subunit beta-2-like 1

Haptoglobin

Homo sapiens

HPT_HUMAN
P00738
45 kDa

HEAT repeat-containing

Homo sapiens

HTR5A_HUMAN
Q86XA9
222 kDa

protein 5A

Heat shock 70 kDa protein
Catarrhini
HSP71_HUMAN
P08107
70 kDa

1A/1B

Heat shock 70 kDa protein 4

Homo sapiens

HSP74_HUMAN
P34932
94 kDa

Heat shock cognate 71 kDa
Eutheria
HSP7C_HUMAN
P11142
71 kDa

protein

Heat shock protein 105 kDa
Hominidae
HS105_HUMAN
Q92598
97 kDa

Heat shock protein beta-1
Catarrhini
HSPB1_HUMAN
P04792
23 kDa

Heat shock protein HSP 90-
Simiiformes
HS90A_HUMAN
P07900
85 kDa

alpha

Heat shock protein HSP 90-

Homo sapiens

HS90B_HUMAN
P08238
83 kDa

beta

Hemoglobin subunit alpha
Hominidae
HBA_HUMAN
P69905
15 kDa

Hemoglobin subunit beta
Homininae
HBB_HUMAN
P68871
16 kDa

Hemoglobin subunit delta

Homo sapiens

HBD_HUMAN
P02042
16 kDa

Hemoglobin subunit gamma-2
Homininae
HBG2_HUMAN
P69892
16 kDa

Hemopexin

Homo sapiens

HEMO_HUMAN
P02790
52 kDa

Heparin cofactor 2

Homo sapiens

HEP2_HUMAN
P05546
57 kDa

Hepatoma-derived growth

Homo sapiens

HDGF_HUMAN
P51858
27 kDa

factor

Hepatoma-derived growth

Homo sapiens

HDGR2_HUMAN
Q7Z4V5
74 kDa

factor-related protein 2

Hepatopoietin PCn127

Homo sapiens

Q1AHP8_HUMAN
Q1AHP8
28 kDa

(+5)

Heterogeneous nuclear

Homo sapiens

ROAA_HUMAN
Q99729
36 kDa

ribonucleoprotein A/B

Heterogeneous nuclear

Homo sapiens

ROA1_HUMAN
P09651
39 kDa

ribonucleoprotein A1

Heterogeneous nuclear
Eutheria
HNRH1_HUMAN
P31943
49 kDa

ribonucleoprotein H

Heterogeneous nuclear
Eutheria
HNRPK_HUMAN
P61978
51 kDa

ribonucleoprotein K

(+1)

Heterogeneous nuclear
Eutheria
HNRPQ_HUMAN
O60506
70 kDa

ribonucleoprotein Q

Heterogeneous nuclear
Eutheria
HNRPR_HUMAN
O43390
71 kDa

ribonucleoprotein R

(+1)

Heterogeneous nuclear

Homo sapiens

HNRPU_HUMAN
Q00839
91 kDa

ribonucleoprotein U

Heterogeneous nuclear
Eutheria
ROA2_HUMAN
P22626
37 kDa

ribonucleoproteins A2/B1

Heterogeneous nuclear
Catarrhini
HNRPC_HUMAN
P07910
34 kDa

ribonucleoproteins C1/C2

Hippocalcin-like protein 1

Homo sapiens

HPCL1_HUMAN
P37235
22 kDa

Histidine-rich glycoprotein

Homo sapiens

HRG_HUMAN
P04196
60 kDa

HMG box transcription factor

Homo sapiens

BBX_HUMAN
Q8WY36
105 kDa

BBX

Hsp70-binding protein 1

Homo sapiens

HPBP1_HUMAN
Q9NZL4
39 kDa

Hydroxymethylglutaryl-CoA

Homo sapiens

HMCS1_HUMAN
Q01581
57 kDa

synthase, cytoplasmic

(+1)

Hypoxanthine-guanine
Catarrhini
HPRT_HUMAN
P00492
25 kDa

phosphoribosyltransferase

Hypoxia up-regulated protein

Homo sapiens

HYOU1_HUMAN
Q9Y4L1
111 kDa

1

Ig alpha-1 chain C region

Homo sapiens

IGHA1_HUMAN
P01876
38 kDa

Ig gamma-2 chain C region

Homo sapiens

IGHG2_HUMAN
P01859
36 kDa

Ig gamma-4 chain C region

Homo sapiens

IGHG4_HUMAN
P01861
36 kDa

Ig heavy chain V-I region

Homo sapiens

HV102_HUMAN
P01743
13 kDa

HG3

Ig kappa chain V-I region

Homo sapiens

KV102_HUMAN
P01594
12 kDa

AU

Ig kappa chain V-II region

Homo sapiens

KV204_HUMAN
P01617
12 kDa

TEW

Ig kappa chain V-III region

Homo sapiens

KV304_HUMAN
P01622
12 kDa

Ti

Ig mu chain C region

Homo sapiens

IGHM_HUMAN
P01871
49 kDa

IGH@ protein

Homo sapiens

Q6GMX6_HUMAN
Q6GMX6
51 kDa

IGK@ protein

Homo sapiens

Q6PIL8_HUMAN
Q6PIL8
26 kDa

IGK@ protein

Homo sapiens

Q6P5S8_HUMAN
Q6P5S8
26 kDa

IGL@ protein

Homo sapiens

Q567P1_HUMAN
Q567P1
25 kDa

Immunoglobulin J chain

Homo sapiens

IGJ_HUMAN
P01591
18 kDa

Importin subunit alpha-2

Homo sapiens

IMA2_HUMAN
P52292
58 kDa

(+1)

Importin subunit beta-1
Hominoidea
IMB1_HUMAN
Q14974
97 kDa

Importin-4

Homo sapiens

IPO4_HUMAN
Q8TEX9
119 kDa

Importin-5

Homo sapiens

IPO5_HUMAN
O00410
124 kDa

Importin-7
Simiiformes
IPO7_HUMAN
O95373
120 kDa

Inositol-3-phosphate synthase

Homo sapiens

INO1_HUMAN
Q9NPH2
61 kDa

1

Insulin-like growth factor 2
Homininae
IF2B2_HUMAN
Q9Y6M1
66 kDa

mRNA-binding protein 2

Insulin-like growth factor-

Homo sapiens

ALS_HUMAN
P35858
66 kDa

binding protein complex acid

(+1)

labile subunit

Integrin beta-1
Homininae
ITB1_HUMAN
P05556
88 kDa

Integrin beta-4

Homo sapiens

ITB4_HUMAN
P16144
202 kDa

Inter-alpha (Globulin)

Homo sapiens

B2RMS9_HUMAN
B2RMS9
103 kDa

inhibitor H4 (Plasma

(+2)

Kallikrein-sensitive

glycoprotein)

Inter-alpha-trypsin inhibitor

Homo sapiens

ITIH1_HUMAN
P19827
101 kDa

heavy chain H1

Inter-alpha-trypsin inhibitor

Homo sapiens

ITIH2_HUMAN
P19823
106 kDa

heavy chain H2

(+1)

Intercellular adhesion

Homo sapiens

ICAM1_HUMAN
P05362
58 kDa

molecule 1

Interleukin enhancer-binding
Eutheria
ILF2_HUMAN
Q12905
43 kDa

factor 2

Interleukin enhancer-binding

Homo sapiens

ILF3_HUMAN
Q12906
95 kDa

factor 3

Iron-responsive element-

Homo sapiens

IREB2_HUMAN
P48200
105 kDa

binding protein 2

Iron-sulfur protein NUBPL

Homo sapiens

NUBPL_HUMAN
Q8TB37
34 kDa

Isocitrate dehydrogenase

Homo sapiens

IDHC_HUMAN
O75874
47 kDa

[NADP] cytoplasmic

Junction plakoglobin
Homininae
PLAK_HUMAN
P14923
82 kDa

Kinesin-1 heavy chain

Homo sapiens

KINH_HUMAN
P33176
110 kDa

Kinesin-associated protein 3
Homininae
KIFA3_HUMAN
Q92845
91 kDa

Kininogen-1

Homo sapiens

KNG1_HUMAN
P01042
72 kDa

(+1)

Kynurenine/alpha-
Catarrhini
AADAT_HUMAN
Q8N5Z0
47 kDa

aminoadipate

aminotransferase,

mitochondrial

Lactoylglutathione lyase

Homo sapiens

LGUL_HUMAN
Q04760
21 kDa

LanC-like protein 2

Homo sapiens

LANC2_HUMAN
Q9NS86
51 kDa

Large neutral amino acids

Homo sapiens

LAT1_HUMAN
Q01650
55 kDa

transporter small subunit 1

(+1)

Large proline-rich protein

Homo sapiens

BAG6_HUMAN
P46379
119 kDa

BAG6

Leucine-rich alpha-2-

Homo sapiens

A2GL_HUMAN
P02750
38 kDa

glycoprotein

Leucine-rich PPR motif-

Homo sapiens

LPPRC_HUMAN
P42704
158 kDa

containing protein,

mitochondrial

Leucine-rich repeat

Homo sapiens

LRRF1_HUMAN
Q32MZ4
89 kDa

flightless-interacting protein

1

Leucine--tRNA ligase,

Homo sapiens

SYLC_HUMAN
Q9P2J5
134 kDa

cytoplasmic

Leukotriene A-4 hydrolase
Homininae
LKHA4_HUMAN
P09960
69 kDa

Liprin-alpha-1

Homo sapiens

LIPA1_HUMAN
Q13136
136 kDa

L-lactate dehydrogenase A

Homo sapiens

LDHA_HUMAN
P00338
37 kDa

chain

L-lactate dehydrogenase B
Catarrhini
LDHB_HUMAN
P07195
37 kDa

chain

Lysine--tRNA ligase

Homo sapiens

SYK_HUMAN
Q15046
68 kDa

Lysophosphatidylcholine

Homo sapiens

PCAT1_HUMAN
Q8NF37
59 kDa

acyltransferase 1

Macrophage mannose

Homo sapiens

MRC1_HUMAN
P22897
166 kDa

receptor 1

(+1)

Macrophage-capping protein

Homo sapiens

CAPG_HUMAN
P40121
38 kDa

Major vault protein

Homo sapiens

MVP_HUMAN
Q14764
99 kDa

Malate dehydrogenase,

Homo sapiens

MDHC_HUMAN
P40925
36 kDa

cytoplasmic

Malate dehydrogenase,

Homo sapiens

MDHM_HUMAN
P40926
36 kDa

mitochondrial

(+1)

Mannosyl-oligosaccharide

Homo sapiens

MOGS_HUMAN
Q13724
92 kDa

glucosidase

(+1)

Microsomal glutathione S-
Homininae
MGST1_HUMAN
P10620
18 kDa

transferase 1

Microtubule-actin cross-

Homo sapiens

MACF1_HUMAN
Q9UPN3
838 kDa

linking factor 1, isoforms

1/2/3/5

Microtubule-associated
Homininae
MAP1B_HUMAN
P46821
271 kDa

protein 1B

Microtubule-associated

Homo sapiens

MAP4_HUMAN
P27816
121 kDa

protein 4

MKI67 FHA domain-

Homo sapiens

MK67I_HUMAN
Q9BYG3
34 kDa

interacting nucleolar

phosphoprotein

Moesin
Catarrhini
MOES_HUMAN
P26038
68 kDa

Monofunctional C1-

Homo sapiens

C1TM_HUMAN
Q6UB35
106 kDa

tetrahydrofolate synthase,

mitochondrial

Msx2-interacting protein

Homo sapiens

MINT_HUMAN
Q96T58
402 kDa

Multifunctional protein

Homo sapiens

PUR6_HUMAN
P22234
47 kDa

ADE2

Myb-binding protein 1A

Homo sapiens

MBB1A_HUMAN
Q9BQG0
149 kDa

Myoferlin

Homo sapiens

MYOF_HUMAN
Q9NZM1
235 kDa

Myosin-10
Homininae
MYH10_HUMAN
P35580
229 kDa

Myosin-6

Homo sapiens

MYH6_HUMAN
P13533
224 kDa

Myosin-9

Homo sapiens

MYH9_HUMAN
P35579
227 kDa

N-acetylmuramoyl-L-alanine

Homo sapiens

PGRP2_HUMAN
Q96PD5
62 kDa

amidase

NACHT, LRR and PYD

Homo sapiens

NALP2_HUMAN
Q9NX02
121 kDa

domains-containing protein 2

NAD(P)H dehydrogenase
Homininae
NQO1_HUMAN
P15559
31 kDa

[quinone] 1

NAD(P)H-hydrate epimerase

Homo sapiens

NNRE_HUMAN
Q8NCW5
32 kDa

NADH-cytochrome b5

Homo sapiens

NB5R3_HUMAN
P00387
34 kDa

reductase 3

NADPH--cytochrome P450

Homo sapiens

NCPR_HUMAN
P16435
77 kDa

reductase

Nascent polypeptide-
Theria
NACA_HUMAN
Q13765
23 kDa

associated complex subunit

alpha

Nebulin

Homo sapiens

NEBU_HUMAN
P20929
773 kDa

Nesprin-2

Homo sapiens

SYNE2_HUMAN
Q8WXH0
796 kDa

Neuroblast differentiation-

Homo sapiens

AHNK_HUMAN
Q09666
629 kDa

associated protein AHNAK

Neurofibromin
Catarrhini
NF1_HUMAN
P21359
319 kDa

Neutral alpha-glucosidase

Homo sapiens

GANAB_HUMAN
Q14697
107 kDa

AB

Niban-like protein 2

Homo sapiens

NIBL2_HUMAN
Q86XR2
77 kDa

Nicotinamide
Homininae
NAMPT_HUMAN
P43490
56 kDa

phosphoribosyltransferase

Nitrilase homolog 1

Homo sapiens

NIT1_HUMAN
Q86X76
36 kDa

N-lysine methyltransferase

Homo sapiens

SETD6_HUMAN
Q8TBK2
53 kDa

SETD6

Nodal modulator 3

Homo sapiens

NOMO3_HUMAN
P69849
134 kDa

(+3)

Nuclear factor erythroid 2-

Homo sapiens

NF2L3_HUMAN
Q9Y4A8
76 kDa

related factor 3

(+1)

Nuclear migration protein

Homo sapiens

NUDC_HUMAN
Q9Y266
38 kDa

nudC

Nucleolar GTP-binding

Homo sapiens

NOG2_HUMAN
Q13823
84 kDa

protein 2

Nucleolar RNA helicase 2

Homo sapiens

DDX21_HUMAN
Q9NR30
87 kDa

Nucleolin

Homo sapiens

NUCL_HUMAN
P19338
77 kDa

Nucleophosmin
Homininae
NPM_HUMAN
P06748
33 kDa

Nucleoside diphosphate
Hominidae
NDKA_HUMAN
P15531
17 kDa

kinase A

Nucleosome assembly
Homininae
NP1L1_HUMAN
P55209
45 kDa

protein 1-like 1

Obg-like ATPase 1
Eutheria
OLA1_HUMAN
Q9NTK5
45 kDa

Ornithine aminotransferase,
Homininae
OAT_HUMAN
P04181
49 kDa

mitochondrial

Oxysterol-binding protein 1

Homo sapiens

OSBP1_HUMAN
P22059
89 kDa

Pachytene checkpoint protein

Homo sapiens

PCH2_HUMAN
Q15645
49 kDa

2 homolog

Peptidyl-prolyl cis-trans
Catarrhini
Q567Q0_HUMAN
Q567Q0
11 kDa

isomerase A

Peptidyl-prolyl cis-trans
Catarrhini
PPIB_HUMAN
P23284
24 kDa

isomerase B

Peroxiredoxin-1
Homininae
PRDX1_HUMAN
Q06830
22 kDa

Peroxiredoxin-2
Catarrhini
PRDX2_HUMAN
P32119
22 kDa

Peroxiredoxin-5,

Homo sapiens

PRDX5_HUMAN
P30044
22 kDa

mitochondrial

Peroxiredoxin-6
Hominidae
PRDX6_HUMAN
P30041
25 kDa

Peroxisomal multifunctional

Homo sapiens

DHB4_HUMAN
P51659
80 kDa

enzyme type 2

Phosphatidylinositol-binding

Homo sapiens

PICAL_HUMAN
Q13492
71 kDa

clathrin assembly protein

Phosphatidylinositol-glycan-

Homo sapiens

PHLD_HUMAN
P80108
92 kDa

specific phospholipase D

Phosphoglucomutase-1

Homo sapiens

PGM1_HUMAN
P36871
61 kDa

Phosphoglycerate kinase 1
Euarchontoglires
PGK1_HUMAN
P00558
45 kDa

Phosphoglycerate mutase 1
Eutheria
PGAM1_HUMAN
P18669
29 kDa

Phosphoribosylglycinamide

Homo sapiens

Q3B7A7_HUMAN
Q3B7A7
108 kDa

formyltransferase,

(+1)

phosphoribosylglycinamide

synthetase,

phosphoribosylaminoimidazole

synthetase

Phosphoserine

Homo sapiens

SERC_HUMAN
Q9Y617
40 kDa

aminotransferase

PIG48

Homo sapiens

Q2TU64_HUMAN
Q2TU64
61 kDa

Pigment epithelium-derived

Homo sapiens

PEDF_HUMAN
P36955
46 kDa

factor

Plasma kallikrein

Homo sapiens

KLKB1_HUMAN
P03952
71 kDa

Plasma protease C1 inhibitor

Homo sapiens

IC1_HUMAN
P05155
55 kDa

Plasminogen

Homo sapiens

PLMN_HUMAN
P00747
91 kDa

Plasminogen activator
Hominoidea
PAIRB_HUMAN
Q8NC51
45 kDa

inhibitor 1 RNA-binding

protein

Platelet-activating factor
Homininae
PA1B3_HUMAN
Q15102
26 kDa

acetylhydrolase IB subunit

gamma

Plectin

Homo sapiens

PLEC_HUMAN
Q15149
532 kDa

Poly [ADP-ribose]

Homo sapiens

PARP1_HUMAN
P09874
113 kDa

polymerase 1

Poly(RC) binding protein 2
Euarchontoglires
Q68Y55_HUMAN
Q68Y55
35 kDa

Poly(rC)-binding protein 1
Eutheria
PCBP1_HUMAN
Q15365
37 kDa

Polyadenylate-binding
Eutheria
PABP1_HUMAN
P11940
71 kDa

protein 1

Polyadenylate-binding

Homo sapiens

PABP4_HUMAN
Q13310
71 kDa

protein 4

(+1)

Polymerase I and transcript

Homo sapiens

PTRF_HUMAN
Q6NZI2
43 kDa

release factor

Prelamin-A/C
Homininae
LMNA_HUMAN
P02545
74 kDa

(+3)

Pre-mRNA-processing factor

Homo sapiens

PR40A_HUMAN
O75400
109 kDa

40 homolog A

Prenylcysteine oxidase 1

Homo sapiens

PCYOX_HUMAN
Q9UHG3
57 kDa

Probable ATP-dependent
Hominidae
DDX17_HUMAN
Q92841
80 kDa

RNA helicase DDX17

Probable ATP-dependent
Hominoidea
DDX6_HUMAN
P26196
54 kDa

RNA helicase DDX6

Probable E3 ubiquitin-protein

Homo sapiens

HERC1_HUMAN
Q15751
532 kDa

ligase HERC1

Probable phospholipid-

Homo sapiens

AT10D_HUMAN
Q9P241
160 kDa

transporting ATPase VD

Probable RNA-binding

Homo sapiens

RBM20_HUMAN
Q5T481
134 kDa

protein 20

Profilin-1
Catarrhini
PROF1_HUMAN
P07737
15 kDa

Programmed cell death

Homo sapiens

PDCD4_HUMAN
Q53EL6
52 kDa

protein 4

Prohibitin
Eutheria
PHB_HUMAN
P35232
30 kDa

Prohibitin-2
Euarchontoglires
PHB2_HUMAN
Q99623
33 kDa

Proliferating cell nuclear

Homo sapiens

Q6FI35_HUMAN
Q6FI35
29 kDa

antigen

Proline-, glutamic acid- and

Homo sapiens

PELP1_HUMAN
Q8IZL8
120 kDa

leucine-rich protein 1

Prolyl 3-hydroxylase 1

Homo sapiens

P3H1_HUMAN
Q32P28
83 kDa

Prolyl endopeptidase

Homo sapiens

PPCE_HUMAN
P48147
81 kDa

Proteasome activator
Hominidae
PSME1_HUMAN
Q06323
29 kDa

complex subunit 1

Proteasome activator
Eutheria
PSME3_HUMAN
P61289
30 kDa

complex subunit 3

Proteasome subunit alpha
Eutheria
PSA1_HUMAN
P25786
30 kDa

type-1

Proteasome subunit alpha
Eutheria
PSA3_HUMAN
P25788
28 kDa

type-3

(+1)

Proteasome subunit alpha
Eutheria
PSA4_HUMAN
P25789
29 kDa

type-4

Proteasome subunit alpha
Eutheria
PSA5_HUMAN
P28066
26 kDa

type-5

(+1)

Proteasome subunit alpha
Eutheria
PSA6_HUMAN
P60900
27 kDa

type-6

Proteasome subunit alpha

Homo sapiens

PSA7_HUMAN
O14818
28 kDa

type-7

Proteasome subunit beta

Homo sapiens

PSB3_HUMAN
P49720
23 kDa

type-3

Proteasome subunit beta
Hominidae
PSB5_HUMAN
P28074
28 kDa

type-5

Protein AHNAK2

Homo sapiens

AHNK2_HUMAN
Q8IVF2
617 kDa

Protein AMBP

Homo sapiens

AMBP_HUMAN
P02760
39 kDa

Protein arginine N-
Catarrhini
ANM5_HUMAN
O14744
73 kDa

methyltransferase 5

Protein diaphanous homolog

Homo sapiens

DIAP1_HUMAN
O60610
141 kDa

1

(+2)

Protein disulfide-isomerase

Homo sapiens

PDIA1_HUMAN
P07237
57 kDa

Protein disulfide-isomerase
Hominidae
PDIA3_HUMAN
P30101
57 kDa

A3

Protein disulfide-isomerase

Homo sapiens

PDIA4_HUMAN
P13667
73 kDa

A4

Protein disulfide-isomerase

Homo sapiens

PDIA6_HUMAN
Q15084
48 kDa

A6

Protein DJ-1
Hominoidea
PARK7_HUMAN
Q99497
20 kDa

Protein FAM49B
Eutheria
FA49B_HUMAN
Q9NUQ9
37 kDa

Protein flightless-1 homolog

Homo sapiens

FLII_HUMAN
Q13045
145 kDa

Protein kinase C inhibitor-2

Homo sapiens

Q8WYJ5_HUMAN
Q8WYJ5
14 kDa

(+1)

Protein kinase, cGMP-
Theria
Q5JP05_HUMAN
Q5JP05
32 kDa

dependent, type I

Protein phosphatase 1G
Homininae
PPM1G_HUMAN
O15355
59 kDa

Protein S100-A10
Theria
S10AA_HUMAN
P60903
11 kDa

Protein SEC13 homolog

Homo sapiens

SEC13_HUMAN
P55735
36 kDa

Protein SET
Homininae
SET_HUMAN
Q01105
33 kDa

Protein transport protein

Homo sapiens

SC24C_HUMAN
P53992
118 kDa

Sec24C

Protein-glutamine gamma-

Homo sapiens

TGM2_HUMAN
P21980
77 kDa

glutamyltransferase 2

Puromycin-sensitive
Homininae
PSA_HUMAN
P55786
103 kDa

aminopeptidase

Putative pre-mRNA-splicing
Homininae
DHX15_HUMAN
O43143
91 kDa

factor ATP-dependent RNA

helicase DHX15

Putative RNA-binding
Simiiformes
LC7L2_HUMAN
Q9Y383
47 kDa

protein Luc7-like 2

PWWP domain-containing

Homo sapiens

MUML1_HUMAN
Q5H9M0
79 kDa

protein MUM1L1

Pyridoxal kinase

Homo sapiens

PDXK_HUMAN
O00764
35 kDa

Pyrroline-5-carboxylate

Homo sapiens

A6NFM2_HUMAN
A6NFM2
33 kDa

reductase

(+3)

Pyruvate kinase isozymes
Homininae
KPYM_HUMAN
P14618
58 kDa

M1/M2

Rab GDP dissociation
Hominoidea
GDIB_HUMAN
P50395
51 kDa

inhibitor beta

(+1)

RAB1B protein

Homo sapiens

Q6FIG4_HUMAN
Q6FIG4
22 kDa

(+1)

Ran-specific GTPase-
Hominidae
RANG_HUMAN
P43487
23 kDa

activating protein

Ras GTPase-activating

Homo sapiens

NGAP_HUMAN
Q9UJF2
129 kDa

protein nGAP

Ras GTPase-activating

Homo sapiens

G3BP1_HUMAN
Q13283
52 kDa

protein-binding protein 1

(+2)

Ras GTPase-activating-like

Homo sapiens

IQGA1_HUMAN
P46940
189 kDa

protein IQGAP1

Ras-related protein Rab-14
Euteleostomi
RAB14_HUMAN
P61106
24 kDa

Ras-related protein Rab-21
Catarrhini
RAB21_HUMAN
Q9UL25
24 kDa

Ras-related protein Rab-5C
Catarrhini
RAB5C_HUMAN
P51148
23 kDa

Ras-related protein Rab-7a
Theria
RAB7A_HUMAN
P51149
23 kDa

Receptor-type tyrosine-

Homo sapiens

PTPRS_HUMAN
Q13332
217 kDa

protein phosphatase S

Replication factor C subunit
Hominoidea
RFC3_HUMAN
P40938
41 kDa

3

Reticulon-4

Homo sapiens

RTN4_HUMAN
Q9NQC3
130 kDa

Rho GTPase-activating

Homo sapiens

RHG32_HUMAN
A7KAX9
231 kDa

protein 32

Rho guanine nucleotide

Homo sapiens

ARHG1_HUMAN
Q92888
102 kDa

exchange factor 1

Ribonuclease inhibitor

Homo sapiens

RINI_HUMAN
P13489
50 kDa

Ribonucleoside-diphosphate

Homo sapiens

RIR2_HUMAN
P31350
45 kDa

reductase subunit M2

Ribosome biogenesis protein

Homo sapiens

BRX1_HUMAN
Q8TDN6
41 kDa

BRX1 homolog

Ribosome-binding protein 1

Homo sapiens

RRBP1_HUMAN
Q9P2E9
152 kDa

ROBO2 isoform a

Homo sapiens

Q19AB5_HUMAN
Q19AB5
153 kDa

(+1)

RPL14 protein

Homo sapiens

Q6IPH7_HUMAN
Q6IPH7
24 kDa

RuvB-like 1
Eutheria
RUVB1_HUMAN
Q9Y265
50 kDa

RuvB-like 2
Catarrhini
RUVB2_HUMAN
Q9Y230
51 kDa

Sacsin

Homo sapiens

SACS_HUMAN
Q9NZJ4
521 kDa

Sarcoplasmic/endoplasmic

Homo sapiens

AT2A2_HUMAN
P16615
115 kDa

reticulum calcium ATPase 2

Sec1 family domain-

Homo sapiens

SCFD1_HUMAN
Q8WVM8
72 kDa

containing protein 1

SEC23-interacting protein

Homo sapiens

S23IP_HUMAN
Q9Y6Y8
111 kDa

Secretory carrier-associated
Homininae
SCAM3_HUMAN
O14828
38 kDa

membrane protein 3

Septin-2
Catarrhini
SEPT2_HUMAN
Q15019
41 kDa

Sequestosome-1

Homo sapiens

SQSTM_HUMAN
Q13501
48 kDa

Serine

Homo sapiens

GLYM_HUMAN
P34897
56 kDa

hydroxymethyltransferase,

(+1)

mitochondrial

Serine palmitoyltransferase 2

Homo sapiens

SPTC2_HUMAN
O15270
63 kDa

Serine/arginine-rich splicing
Amniota
SRSF3_HUMAN
P84103
19 kDa

factor 3

Serine/threonine-protein

Homo sapiens

NEK9_HUMAN
Q8TD19
107 kDa

kinase Nek9

Serine/threonine-protein

Homo sapiens

OXSR1_HUMAN
O95747
58 kDa

kinase OSR1

Serine/threonine-protein

Homo sapiens

PAK2_HUMAN
Q13177
58 kDa

kinase PAK 2

Serine/threonine-protein
Simiiformes
2AAA_HUMAN
P30153
65 kDa

phosphatase 2A 65 kDa

regulatory subunit A alpha

isoform

Serine/threonine-protein

Homo sapiens

PTPA_HUMAN
Q15257
41 kDa

phosphatase 2A activator

Serine/threonine-protein
Theria
PP2AA_HUMAN
P67775
36 kDa

phosphatase 2A catalytic

(+1)

subunit alpha isoform

Serine--tRNA ligase,

Homo sapiens

SYSC_HUMAN
P49591
59 kDa

cytoplasmic

(+1)

Serotransferrin

Homo sapiens

TRFE_HUMAN
P02787
77 kDa

(+1)

Serpin B6

Homo sapiens

SPB6_HUMAN
P35237
43 kDa

Serum albumin

Homo sapiens

ALBU_HUMAN
P02768
69 kDa

Serum albumin

Bos taurus

ALBU_BOVIN
P02769
69 kDa

Serum amyloid A-4 protein

Homo sapiens

SAA4_HUMAN
P35542
15 kDa

Serum amyloid P-component

Homo sapiens

SAMP_HUMAN
P02743
25 kDa

Serum

Homo sapiens

PON1_HUMAN
P27169
40 kDa

paraoxonase/arylesterase 1

SH2 domain-containing

Homo sapiens

SHE_HUMAN
Q5VZ18
54 kDa

adapter protein E

SH3 domain-containing

Homo sapiens

SH3K1_HUMAN
Q96B97
73 kDa

kinase-binding protein 1

(+1)

Sialic acid synthase
Homininae
SIAS_HUMAN
Q9NR45
40 kDa

Signal peptide, CUB and

Homo sapiens

SCUB2_HUMAN
Q9NQ36
110 kDa

EGF-like domain-containing

protein 2

Signal recognition particle 54
Eutheria
SRP54_HUMAN
P61011
56 kDa

kDa protein

Signal recognition particle 72

Homo sapiens

SRP72_HUMAN
O76094
75 kDa

kDa protein

Small nuclear
Eutheria
RSMB_HUMAN
P14678
25 kDa

ribonucleoprotein-associated

(+4)

proteins B and B′

Sodium/potassium-

Homo sapiens

AT1A1_HUMAN
P05023
113 kDa

transporting ATPase subunit

alpha-1

Solute carrier family 2,
Homininae
GTR1_HUMAN
P11166
54 kDa

facilitated glucose transporter

(+1)

member 1

Sorbitol dehydrogenase

Homo sapiens

DHSO_HUMAN
Q00796
38 kDa

Sorting nexin-6

Homo sapiens

SNX6_HUMAN
Q9UNH7
47 kDa

SP-A receptor subunit SP-

Homo sapiens

Q5QD01_HUMAN
Q5QD01
181 kDa

R210 alphaS

(+1)

Spectrin alpha chain, brain

Homo sapiens

SPTA2_HUMAN
Q13813
285 kDa

Spectrin beta chain, brain 1
Homininae
SPTB2_HUMAN
Q01082
275 kDa

Spectrin repeat containing,

Homo sapiens

Q5JV23_HUMAN
Q5JV23
1005 kDa

nuclear envelope 1

(+1)

Sphingosine-1-phosphate

Homo sapiens

SGPL1_HUMAN
O95470
64 kDa

lyase 1

Spliceosome RNA helicase
Theria
DX39B_HUMAN
Q13838
49 kDa

DDX39B

Splicing factor 3B subunit 3
Eutheria
SF3B3_HUMAN
Q15393
136 kDa

Splicing factor, proline- and
Eutheria
SFPQ_HUMAN
P23246
76 kDa

glutamine-rich

(+1)

Staphylococcal nuclease

Homo sapiens

SND1_HUMAN
Q7KZF4
102 kDa

domain-containing protein 1

STIP1 protein

Homo sapiens

Q3ZCU9_HUMAN
Q3ZCU9
68 kDa

Stomatin-like protein 2
Hominidae
STML2_HUMAN
Q9UJZ1
39 kDa

Stress-70 protein,

Homo sapiens

GRP75_HUMAN
P38646
74 kDa

mitochondrial

Structural maintenance of

Homo sapiens

SMHD1_HUMAN
A6NHR9
226 kDa

chromosomes flexible hinge

domain-containing protein 1

Superoxide dismutase [Cu—Zn]
Homininae
SODC_HUMAN
P00441
16 kDa

Talin-1

Homo sapiens

TLN1_HUMAN
Q9Y490
270 kDa

T-complex protein 1 subunit
Catarrhini
TCPA_HUMAN
P17987
60 kDa

alpha

T-complex protein 1 subunit
Catarrhini
TCPB_HUMAN
P78371
57 kDa

beta

T-complex protein 1 subunit
Hominoidea
TCPD_HUMAN
P50991
58 kDa

delta

T-complex protein 1 subunit
Catarrhini
TCPE_HUMAN
P48643
60 kDa

epsilon

T-complex protein 1 subunit
Hominoidea
TCPZ_HUMAN
P40227
58 kDa

zeta

Testin
Hominidae
TES_HUMAN
Q9UGI8
48 kDa

Thioredoxin reductase 1,

Homo sapiens

TRXR1_HUMAN
Q16881
71 kDa

cytoplasmic

Thioredoxin-dependent
Homininae
PRDX3_HUMAN
P30048
28 kDa

peroxide reductase,

mitochondrial

THO complex subunit 4

Homo sapiens

THOC4_HUMAN
Q86V81
27 kDa

Threonine--tRNA ligase,

Homo sapiens

SYTC_HUMAN
P26639
83 kDa

cytoplasmic

Tight junction protein ZO-2

Homo sapiens

ZO2_HUMAN
Q9UDY2
134 kDa

Titin

Homo sapiens

TITIN_HUMAN
Q8WZ42
3816 kDa

Transcription elongation

Homo sapiens

ELOA1_HUMAN
Q14241
90 kDa

factor B polypeptide 3

Transcription factor p65
Homininae
TF65_HUMAN
Q04206
60 kDa

Transcription intermediary
Homininae
TIF1B_HUMAN
Q13263
89 kDa

factor 1-beta

Transferrin receptor protein 1

Homo sapiens

TFR1_HUMAN
P02786
85 kDa

Transgelin-2
Catarrhini
TAGL2_HUMAN
P37802
22 kDa

Transitional endoplasmic
Euarchontoglires
TERA_HUMAN
P55072
89 kDa

reticulum ATPase

Transketolase

Homo sapiens

TKT_HUMAN
P29401
68 kDa

Translational activator GCN1

Homo sapiens

GCN1L_HUMAN
Q92616
293 kDa

Translin
Euarchontoglires
TSN_HUMAN
Q15631
26 kDa

Translocon-associated

Homo sapiens

SSRA_HUMAN
P43307
32 kDa

protein subunit alpha

Transmembrane emp24

Homo sapiens

TMEDA_HUMAN
P49755
25 kDa

domain-containing protein 10

Transportin-1
Homininae
TNPO1_HUMAN
Q92973
102 kDa

Trifunctional enzyme subunit

Homo sapiens

ECHA_HUMAN
P40939
83 kDa

alpha, mitochondrial

Triosephosphate isomerase
Homininae
TPIS_HUMAN
P60174
31 kDa

tRNA (cytosine(34)-C(5))-

Homo sapiens

NSUN2_HUMAN
Q08J23
86 kDa

methyltransferase

tRNA-splicing ligase RtcB
Eutheria
RTCB_HUMAN
Q9Y3I0
55 kDa

homolog

Tropomodulin-3

Homo sapiens

TMOD3_HUMAN
Q9NYL9
40 kDa

Tropomyosin 3
Eutheria
Q5VU58_HUMAN
Q5VU58
29 kDa

Tryptophan--tRNA ligase,
Hominidae
SYWC_HUMAN
P23381
53 kDa

cytoplasmic

Tubulin alpha-1C chain
Hominidae
TBA1C_HUMAN
Q9BQE3
50 kDa

Tubulin alpha-4A chain
Eutheria
TBA4A_HUMAN
P68366
50 kDa

Tubulin beta chain
Amniota
TBB5_HUMAN
P07437
50 kDa

Tubulin beta-4B chain
Theria
TBB4B_HUMAN
P68371
50 kDa

(+1)

Tubulin beta-6 chain
Hominoidea
TBB6_HUMAN
Q9BUF5
50 kDa

Tyrosine-protein phosphatase

Homo sapiens

PTN13_HUMAN
Q12923
277 kDa

non-receptor type 13

U5 small nuclear
Hominidae
U520_HUMAN
O75643
245 kDa

ribonucleoprotein 200 kDa

helicase

Ubiquitin carboxyl-terminal
Homininae
UBP5_HUMAN
P45974
96 kDa

hydrolase 5

Ubiquitin carboxyl-terminal

Homo sapiens

UBP8_HUMAN
P40818
128 kDa

hydrolase 8

Ubiquitin carboxyl-terminal
Hominidae
UCHL1_HUMAN
P09936
25 kDa

hydrolase isozyme L1

Ubiquitin thioesterase
Eutheria
OTUB1_HUMAN
Q96FW1
31 kDa

OTUB1

Ubiquitin-60S ribosomal
Tetrapoda
RL40_HUMAN
P62987
15 kDa

protein L40

(+3)

Ubiquitin-associated protein

Homo sapiens

UBP2L_HUMAN
Q14157
115 kDa

2-like

Ubiquitin-like modifier-

Homo sapiens

UBA1_HUMAN
P22314
118 kDa

activating enzyme 1

UDP-glucose 6-
Hominidae
UGDH_HUMAN
O60701
55 kDa

dehydrogenase

UDP-glucose: glycoprotein

Homo sapiens

UGGG1_HUMAN
Q9NYU2
177 kDa

glucosyltransferase 1

UDP-N-acetylhexosamine

Homo sapiens

UAP1_HUMAN
Q16222
59 kDa

pyrophosphorylase

UMP-CMP kinase

Homo sapiens

KCY_HUMAN
P30085
22 kDa

(+2)

Unc-45 homolog A

Homo sapiens

A8K6F7_HUMAN
A8K6F7
102 kDa

(C. elegans), isoform CRA_a

(+1)

Unconventional myosin-Ic

Homo sapiens

MYO1C_HUMAN
O00159
122 kDa

Unconventional myosin-Ie

Homo sapiens

MYO1E_HUMAN
Q12965
127 kDa

Unconventional myosin-VI

Homo sapiens

MYO6_HUMAN
Q9UM54
150 kDa

Uridine 5′-monophosphate

Homo sapiens

UMPS_HUMAN
P11172
52 kDa

synthase

Uridine phosphorylase 1
Homininae
UPP1_HUMAN
Q16831
34 kDa

UTP--glucose-1-phosphate
Hominidae
UGPA_HUMAN
Q16851
57 kDa

uridylyltransferase

UV excision repair protein
Homininae
RD23B_HUMAN
P54727
43 kDa

RAD23 homolog B

Example 11—Summary of the Methods of the Present Disclosure Versus Commercially Available Kits

As can be seen from the results above, the methods of the present disclosure provide a mechanism to isolate biologically relevant circulating cell free nucleic acids along with associated cellular components, such as lipids and polypeptides. Both the nucleic acid and cellular components can be used to determine the condition of a subject and to analyze a variety of biomarkers. Furthermore the methods of the present disclosure selectively isolate active genomic regions, selecting for the most relevant nucleic acid targets for analysis. In addition to increased purity and yield, the methods of the present disclosure also use significantly smaller volumes of sample starting material for the analysis. In summary, the methods of the present disclosure provide significant advantage over the methods known in the prior art. A comparison of the characteristic of the methods of the present disclosure versus the prior art is provided in Table 11.

Methods of the Prior

Art
Present Disclosure

Main Principle
Random, blinded
Selective enrichment of

isolation of nucleic acid
lipid/polypeptide associated

nucleic acid complexes

(active genomic regions)

Genomic
Poor-Fair
Excellent

Representation

of Active Gene

(eg, oncogenes)

Starting
Large Volume
Very Small Volume

Material

(eg, plasma)

Yield
Low
High

Functionality
No
Yes

Studies

ID Associated
No
Yes

Biomarkers

Sensitivity/
Low (solely depends on
High

Specificity
extraction efficiency)

METHODS OF ISOLATION OF CELL FREE COMPLEXES AND CIRCULATING CELL-FREE NUCLEIC ACID

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

International Classifications

Abstract

Description

Claims

PCT Information

Provisional Applications (1)