Patent Application
20050032116
The present invention is related to the field of proteomics and diagnostics and generally relates to the qualitative and quantitative characterization of insulin-like growth factors present in humans by use of mass spectrometry. More specifically, the present invention encompasses affinity capture methods and devices used for the selective retrieval of insulin like growth factor 1 (IGF-1) and insulin like growth factor 2 (IGF-2) from human biological fluids prior to mass spectrometric interrogation. The methods and devices can be used in stand-alone application with mass spectrometry, as in the case of Mass Spectrometric Immunoassay (MSIA), or can include methods of optical sensing during biomolecular recognition events, as in the case of Biomolecular Interaction Analysis Mass Spectrometry (BIA/MS). These methods and devices, which target IGF-1 or IGF-2 separately in individual assays, or both species in a simultaneous assay, find application in the clinical and diagnostic monitoring of the growth factors for the presence of interacting partners, qualitative changes brought on by genetic or posttranslational causes, or quantitave modulation due to disease or ailment.
With the recent first draft completion of the human genome, much attention is now shifting to the field of proteomics, where gene products (proteins), their variants, interacting partners and the dynamics of their regulation and processing are the emphasis of study. Such studies are essential in understanding, for example, the mechanisms behind genetic/environmentally induced disorders or the influences of drug mediated therapies, and are potentially becoming the underlying foundation for further clinical and diagnostic analyses. Critical to these studies is the ability to qualitatively determine specific variants of whole proteins (i.e., splice variants, point mutations and posttranslationally modified versions) and the ability to view their quantitative modulation.
Growth factors, in particular, are the subject of much study with regard to relating physiological changes (i.e., qualitative and quantitative modulation) to disease. Specifically, the insulin like growth factors 1 and 2 (IGF-1 and IGF-2), which are members of an important network of proteins that regulate metabolic, growth, and other cellular processes and activities, have been linked to abnormal growth, prostate cancer and breast cancer. Primarily synthesized in the liver, the IGFs circulate in serum in a form of protein complexes, bound to IGF-binding proteins (IGFBP). Less than 1% of the IGFs circulate in free, unassociated form. The binding to the IGFBPs increases the half-life of IGFs in blood, whereas the physiological role of the free IGF has not yet been determined. Structurally, IGF-1 and IGF-2 share 62% amino acid sequence homology, and there is 40% homology between the IGFs and proinsulin.
Immunoassays (ELISA, radio, or chemiluminescence) are generally used for assaying IGFs in plasma/serum. Because the concentration of free IGFs in serum samples can increase upon storage (due to proteases-induced release of the bound IGFs), determination of the total IGF is preferred in clinical research and practice. Acid ethanol extraction is commonly used to release the bound IGFs prior to assaying, although additional steps are often required to minimize the IGFBPs interference. IGFs measurements are routinely performed using commercially available immunoassays, and recently studies on large populations have yielded important correlations between increased IGF concentrations and the risk of cancer.
Although the conventional immunoassay approaches have found considerable use in the quantitative monitoring of the growth factors, they suffer from a common fault of all immunological assays that rely on the indirect detection of the species under investigation; that being the inability to readily differentiate between variants of the same protein. With regard to human beings, there are several possible causes for the presence of multiple and variable species of the same protein in individuals. These causes include, genetic heterozygosity, translational splice variation and/or variable posttranslational modifications. The two former causes require that any quantitative assay be accompanied by a second assay that is able to qualify (i.e., either confirm the wild-type or determine a mutant) gene sequence. Likewise, the latter cause requires an additional qualitative assay to confirm that the protein under investigation is in fact the “correct” form, i.e., of wild-type posttranslational modification. Thus, for absolute certainty, any immunological assay that utilizes indirect means of detection (e.g., secondary antibody conjugated to a fluorescent or radioactive reporter) must be accompanied by a second qualitative analysis able to unambiguously confirm or identify the exact (not presumed) protein species under investigation. Because of strict biological function—structure relationships, quantitative assays not accompanied by corresponding rigorous qualitative assay can in the least be erroneous, and, at worst, meaningless.
Moreover, there are several real-life challenges inherent to the analysis of the IGFs, and of all proteins in general. Foremost is the fact that any protein considered relevant enough to be analyzed resides in vivo in a complex biological environment or media. The complexity of these biological media present a challenge in that, oftentimes, a protein of interest is present in the media at relatively low levels and is essentially masked from analysis by a large abundance of other biomolecules, e.g., proteins, nucleic acids, carbohydrates, lipids and the like. In other instances, proteins are complexed tightly with other biomolecules that might interfere with their analysis. In order to analyze proteins of interest from- and in- their native environment, assays capable of assessing proteins present in a variety of biological fluids, both qualitatively and quantitatively, are needed. These assays must: 1) Be able to selectively retrieve and concentrate specific proteins/biomarkers from biological fluid for subsequent high-performance analyses, 2) Be able to quantify targeted proteins, 3) Be able to recognize variants of targeted proteins (e.g., splice variants, point mutations and posttranslational modifications) and to elucidate their nature, and 4) Be capable of analyzing for, and identifying, ligands interacting with targeted proteins.
Thus, there is a pressing need for new and improved technologies able to characterize insulin-like growth factors, both qualitatively and quantitatively, in a single assay. Likewise, there exists a pressing need for techniques that are able to readily study both unbound (i.e., free in solution) and bound (i.e., complexed with IGFBP and other proteins) insulin-like growth factors and associated components for use in the study of the biophysical properties of wild-type and variant forms of the growth factors. Two protein mass spectrometry techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry, offer the particular advantages of detecting multiple proteins in the same analysis and being able to differentiate between mass-shifted variant forms of the same protein. Mass resolution of related protein species also allows mass-shifted variants of a target protein to be intentionally incorporated into the analysis for use as internal reference standards for quantitative analysis. In this manner, affinity capture assays can be designed where a single pan-antibody is used to retrieve all protein variants (and in vivo assembled complexes) from a biological fluid, upon which each variant or component is detected during mass spectrometry at a unique and characteristic molecular mass. Two recently developed technologies that use this affinity retrieval procedure in a combination with mass spectrometry for detection and characterization of proteins from complex biological fluids are Mass Spectrometric Immunoassay (MSIA) (Nelson, R. W., Krone, J. R., Bieber, A. L. and Williams, P. (1995) Anal. Chem. 67, 1153-1158; Niederkofler, E. E., Tubbs, K. A., Gruber, K., Nedelkov, D., Kiernan, U. A., Williams, P. and Nelson, R. W. (2001) Anal. Chem. 73, 3294-3299; Kiernan, U., Tubbs, K., Nedelkov, D., Niederkofler, E. and Nelson, R. (2002) Biochem. Biophys. Res. Commun. 297, 401; Tubbs, K. A., Nedelkov, D. and Nelson, R. W. (2001) Anal. Biochem. 289, 26-35)—an assay that is used for the unambiguous detection and rigorous quantification of polypeptides/proteins retrieved from complex biological systems, and Biomolecular Interaction Analysis Mass Spectrometry (BIA/MS) (Krone, J. R., Nelson, R. W., Dogruel, D., Williams, P. and Granzow, R. (1997) Anal. Biochem. 244, 124-32; Nelson, R. W., Krone, J. R. and Jansson, O. (1997) Anal. Chem. 69, 4363-8; Nelson, R. W., Nedelkov, D. and Tubbs, K. A. (2000) Anal. Chem. 72, 404A-411A; Nedelkov, D. and Nelson, R. W. (2000) J. Mol. Recogn. 13, 140-145; Nedelkov, D. and Nelson, R. W. (2001) Am. J. Kidney Dis. 38, 481-7)—which combines Surface Plasmon Resonance (SPR) quantification with mass spectrometry.
For the foregoing reasons, there is a need for MSIA and BIA/MS devices, kits, methods and protocols for the rapid and efficient qualitative and quantitative characterization of insulin-like growths factors, their phenotypic variants and their in vivo binding components.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the invention.
It is an object of the present invention to devise MSIA and BIA/MS methods that prepare IGF 1 and 2, in micro-sample form, directly from biological fluid to be used in detecting and quantifying the growth factors present in human plasma and serum.
It is another object of the present invention to construct devices, in the form of pipettor tips containing porous solid supports that are constructed, covalently derivatized with affinity ligand (termed MSIA-Tips), that extract IGF-1 and IGF-2, and their variants, from various biological fluids by repeatedly flowing the fluids through the MSIA-Tips.
It is yet another objective of the present invention to incorporate internal references species (IRS)—mass-shifted variants of the insulin-like growth factors—into analytical samples for co-extraction with the IGFs (in order to normalize sample extractions and data acquisition) for quantification of the growth factors.
It is still a further objective of the present invention to use either MSIA or BIA/MS in the protein phenotyping of individuals by detecting and identifying point mutations or posttranslational variants of the IGFs.
Yet another objective of the present invention is the development of multi-analyte assays capable of simultaneously characterizing both IGF-1 and IGF-2 in a single analysis.
It is still another objective of the present invention to use BIA/MS for both the optical and mass spectrometric characterization of insulin-like growth factors in either their native, in vivo environment or in denaturing conditions
A further object of the present invention enables useful product kits for the characterization of insulin-like growth factors directly from biological fluids for linkage and correlation to disease.
The present invention includes the ability to selectively retrieve and concentrate insulin-like growth factors from biological fluid for subsequent high-performance analyses (e.g. MALDI-TOF MS), the ability to identify targeted biomolecules, the ability to quantify targeted biomolecules, the ability to recognize variants of targeted biomolecules (e.g., splice variants, point mutations and posttranslational modifications) and to elucidate their nature, and the capability to analyze for, and identify, ligands interacting with targeted biomolecules. The invention itself, both as to its structure and its operation together with the additional objects and advantages thereof will best be understood from the following description of the preferred embodiment of the present invention when read in conjunction with the accompanying drawings. The preferred embodiment of the invention is described bellow in the Drawings and Description of Preferred Embodiments. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s). The foregoing description of a preferred embodiment and best mode of the invention known to the applicant at the time of filing the application has been presented and is intended for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in the light of the above teachings. The embodiment was chosen and described in order to best explain the principles of the invention and its practical application and to enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.
The present invention provides for methods, devices and kits for the BIA/MS analysis of insulin-like growth factors, their variants and binding partners present in various biological fluids.
Another embodiment of the present invention provides for methods used in the comparative and rigorous SPR quantification of IGFs and their variants present in various biological fluids.
Still another embodiment of the present invention provides for methods, devices and kits to be used in the MSIA analysis of insulin-like growth factors and their variants present in various biological fluids.
Yet another embodiment of the present invention provides for methods used in the MSIA quantification of IGFs and their variants present in various biological fluids.
Still yet another embodiment of the present invention enables the simultaneous detection and characterization of IGF-1 and IGF-2 in a single MSIA or BIA/MS assay.
Yet another embodiment of the present invention provides for the use of MSIA or BIA/MS in screening of individuals or large populations for IGFs and variants present in various biological fluids.
Specific embodiments in accordance with the present invention will now be described in detail using the following lexicon. These examples are intended to be illustrative, and the invention is not limited to the materials, methods or apparatus set forth in these embodiments.
As used herein, “MSIA-Tips” refers to a pipettor tip containing an affinity reagent.
As used herein, “affinity reagent” refers to a contiguous (formed/molded), porous, high surface area base support containing a low dead-volume (e.g. <2 μL of unused volume) to which affinity ligands are immobilized. The composition of the base support may be, but is not limited to, glasses, silica glasses, silica, silicon, plastics, polymers, metals, or any combination of these materials and the like. Affinity ligands are immobilized to the base support through the process of chemical activation.
As used herein “chemically activate” refers to the process of exposing the affinity reagent to chemicals in order to subsequently attach tethering linkers and/or affinity ligands. Compounds able to activate affinity reagents may be, but are not limited to organic or inorganic reagents. Often, it is advantageous to activate the affinity reagent base support using multiple steps including the use of a tethering linker. As used herein, “tethering linker” refers to compounds intermediate to the base support and the affinity ligand that exhibit the desirable characteristics of being able to be derivatized with high densities of affinity ligand and showing low binding of non-specified compounds. The tethering linker may be intrinsically active or require activation for attachment. Suitable tethering compounds include, but are not limited to, homo/hetero functional organics, natural and synthetic polymers, and biopolymers.
As used herein, “affinity ligand” refers to atomic or molecular species having an affinity towards analytes present in biological mixtures. Affinity ligands may be organic, inorganic or biological by nature, and can exhibit broad (targeting numerous analytes) to narrow (target a single analyte) specificity. Examples of affinity ligands include, but are not limited to, receptors, antibodies, antibody fragments, synthetic paratopes, enzymes, proteins, multi-subunit protein receptors, mimics, chelators, nucleic acids, and aptamers.
As used herein, “analyte” refers to molecules of interest present in a biological sample. Analytes may be, but are not limited to, nucleic acids, DNA, RNA, peptides, polypeptides, proteins, antibodies, protein complexes, carbohydrates or small inorganic or organic molecules having biological function. Analytes may naturally contain sequences, motifs or groups recognized by the affinity ligand or may have these recognition moieties introduced into them via chemical or enzymatic processes.
As used herein, “biological fluid” refers to a fluid or extract having a biological origin. Biological fluid may be, but are not limited to, cell extracts, nuclear extracts, cell lysates or biological products used to induce immunity or substances of biological origin such as excretions, blood, sera, plasma, urine, sputum, tears, feces, saliva, membrane extracts, and the like.
As used herein, “internal reference standard” (IRS) refers to analyte species that are modified (either naturally or intentionally) to result in a molecular weight shift from targeted analytes and their variants. The IRS can be endogenous in the biological fluid or introduced intentionally. The purpose of the IRS is that of normalizing all extraction, rinsing, elution and mass spectrometric steps for the purpose of quantifying targeted analytes and/or variants.
As used herein, “posttranslational modification” refers to any polypeptide alteration that occurs after synthesis of the chain. Posttranslational modifications may be, but are not limited to, glycosylations, phosphorylations, and the like.
As used herein, “mass spectrometer” refers to a device able to volatilize/ionize analytes to form vapor-phase ions and determine their absolute or relative molecular masses. Suitable forms of volatilization/ionization are laser/light, thermal, electrical, atomized/sprayed and the like or combinations thereof. Suitable forms of mass spectrometry include, but are not limited to, Matrix Assisted Laser Desorption/Time of Flight Mass Spectrometry (MALDI-TOF MS), electrospray (or nanospray) ionization (ESI) mass spectrometry, or the like or combinations thereof.
The following examples illustrate the analysis of IGF-1 and IGF-2 via BIA/MS and MSIA.
In its core, BIA/MS is a synergy of two individual technologies: surface plasmon resonance (SPR) sensing and matrix-assisted laser desorption/ionization time-of-flight. (MALDI-TOF) mass spectrometry (
Following antibody immobilization as described in EXAMPLE 2, a 50 μL aliquot of fresh, 50-fold diluted human plasma was injected over the antibody-derivatized FC1 and FC2 surfaces (
In order to eliminate the possibility of cross-walking between the adjacent flow cells, two additional CM5 chips was utilized: a single flow cell on the first chip was derivatized with anti-IGF-1, and one flow cell on the second chip was derivatized with anti-IGF-2. A 50 μL aliquot of fresh human plasma, diluted 10-fold, was injected over both chips in two separate experiments (sensorgrams not shown), and the chips were undocked and analyzed using MALDI-TOF MS. The resulting mass spectra are shown in
In order to more substantially demonstrate the retrieval of the protein complex, fresh human plasma was treated with several detergents to possibly disrupt the protein complex and release its constituent proteins. For the first sample, 20 μL of pure plasma (undiluted) was mixed with 20 μL of 0.5% SDS solution, incubated 30 min at room temperature, and further diluted with 160 μL of HBS-EP buffer to yield a plasma sample diluted 10-fold in buffer and 0.05% SDS. Another sample of plasma (10-fold diluted) was prepared in HBS-EP buffer containing 0.1% Tween 20. These two samples, along with a non-treated plasma control sample (10-fold diluted in HBS-EP) were injected in 10 μL aliquots over anti-IGF-1 and IGF-2 derivatized surfaces on a new CM5 sensor chip. The resulting sensorgrams are shown in
The general MSIA approach is shown graphically in
MSIA-Tips targeting IGF 1 and IGF 2 were prepared by covalently linking anti-IGF (1 or 2) antibodies onto frits contained within pipettor tip barrels. The frits were produced in bulk by loading soda lime glass beads into stainless steel annealing molds and baked to form a solid, yet porous frit. The frits were then removed and acid conditioned prior to a 12-hour treatment with 10% aminopropyl triethoxysilane. The amine-functionalized frits were then equilibrated in a phosphate buffer, after which it was replaced with a mixture of 15-kDa molecular mass carboxymethyl dextran (CMD), and N, N′-carbonyl diimidazole (CDI) to produce frits with surfaces covered with carboxyl groups. The carboxyl groups were activated, prior to antibody coupling, by vigorously rinsing away any free CMD with phosphate buffer and activating the carboxyl surface with an additional volume of CDI. The activated frits were loaded into wide-bore P-200 pipette tips and the tips were subsequently attached to a 96-format robotic pipetting workstation. In-robotic antibody coupling was performed by first flowing 100 μL of anti-IGF (1 or 2) antibody solution (0.1 mg/mL in 10 mM sodium acetate, pH 4.8) through the frits for approximately 40 minutes (by aspirating and dispensing 50 μL volumes). The remaining active sites of the frit were blocked with ethanolamine (1M, pH 8.5) and the tips were equilibrated in HBS buffer prior to their use. This process yielded affinity tips targeting the IGFs, which were found to be stable and active for a period of at least one month following antibody coupling (by storing at 4° C. in saline buffer).
Individual samples for MSIA were prepared by mixing 40-μL aliquots of whole plasma with 60 μL of HEPES buffered saline solution (HBS) and 60 μL of a 0.05% SDS (w/v). The mixture was given adequate time (˜15 minutes) to disrupt all in vivo bound IGFs from their protein complexes, whereupon an additional 840 μL of HBS buffer was added to the solution. IGF-1 or IGF-2 was selectively extracted from the diluted, SDS-treated plasma by repeatedly aspirating and then expelling (˜50 times) 200 μL aliquots of solution through MSIA-Tips, derivatized with either anti-IGF-1 or anti-IGF-2 antibody. After extraction, residual, non-targeted species were removed from the MSIA-Tips by rinsing with: 5×200 μL HBS; 3×200 μL H2O; 3×200 μL 20:80 ACN:H2O; and 3×200 μL H2O. Retained species were eluted from the MSIA-Tips and prepared for MALDI-TOF MS by drawing ˜4 μL of the MALDI matrix α-cyano-4-hydroxycinnamic acid (ACCA; dissolved in 1:2 ACN:H2O, 0.03% TFA) into the tip and expelling/depositing the matrix/eluate mixture directly onto a MALDI-TOF MS target. MALDI-TOF MS then proceeded as generally practiced.
MSIA-Tips were prepared as described in EXAMPLE 7, with the exception of using a mixture of anti-IGF-1 and anti-IGF-2 IgG in place of the singe antibody solutions. Subsequently, plasma, prepared as described in EXAMPLE 8, was analyzed for both IGF-1 and IGF-2 in a single analysis by using the MSIA-Tips that target both of the growth hormones.
The IGF-1 MSIA analyses were made rigorously quantitative by inclusion of an internal reference standard (IRS) into the analysis, and the generation of a calibration curve (working curve) that equates concentration (of endogenous IGF-1) with relative signal intensity (human IGF-1/IRS). Because of similarity in amino acid sequence, cross-reactivity with anti-human IGF-11 antibody, and a resolvable mass difference, rat IGF-1 (rIGF-1) present in rat plasma was used as an IRS.
Samples for generating a quantitative calibration curve for hIGF-1 were prepared as described in EXAMPLE 8, except now each sample included a 20 μL aliquot of rat plasma (note: the initial 60 μL aliquot of HBS was reduced to 40 μL in this procedure) and the 40 μL human plasma sample was substituted with a 40 μL aliquot of purified hIGF-1 standard). Eight hIGF-1 standards at (equivalent plasma) concentrations ranging from 0.008 to 1 μg/mL were prepared for analysis. Both hIGF-1 and rIGF-1 were co-extracted from the samples using anti-IGF-1 MSIA-Tips prepared as described in EXAMPLE 7 and prepared for mass analysis as described in EXAMPLE 8.
The quantitative IGF-1 MSIA was applied to eight individuals (3 females and 5 males; age range 28-46 years old) to determine to concentration of IGF-1 present in plasma.
The present invention and the results shown in the Figures and Examples clearly demonstrate the usefulness of BIA/MS and MSIA in the analysis of insulin-like growth factors and their variants present in various biological fluids as well as the need for methods, devices and kits to expedite and enable the use of BIA/MS and MSIA in the analysis of large numbers of individuals.
This application claims the benefit of, and priority to, provisional application Ser. No. 60/439,110, filed Jan. 10, 2003, which application is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60439110 | Jan 2003 | US |
