Patent Application
20230035606
Cardiovascular diseases account for approximately one out of every four deaths in the United States, with an estimated healthcare burden of over $200 billion per year. Early and accurate diagnosis of heart failure enables successful patient outcomes and reduces the need for excessive and costly testing. Cardiac troponin I (cTnI) is clinically recognized as a sensitive and specific protein biomarker for acute coronary syndrome because it has an amino acid sequence specific to cardiac tissue and is released into the bloodstream following cardiac injury (Missov et al., 1997, Circulation, 96:2953-2958). Additionally, increased circulating cTnI concentration is correlated to the onset of cardiac damage (Thygesen et al., 2018, Journal of the American College of Cardiology, 25285; Westermann et al., 2017, Nature Reviews Cardiology, 14: 472; and Antman et al., 1996, New England Journal of Medicine 335: 1342-1349).
For these reasons, cTnI is a gold-standard biomarker used in the clinical evaluation of myocardial injury. Circulating cTnI in the blood exists in low abundance and in myriad proteoforms (Smith et al., 2013, Nature Methods, 10:186) (e.g., phosphorylated, truncated, acetylated, and/or oxidized forms of the protein) that are known to reflect pathophysiological processes (Bates et al., 2010, Clinical Chemistry, 56:952; Madsen Lene et al., 2006, Circulation Research, 99:1141-1147; and Soetkamp et al., 2017, Expert Review of Proteomics 14: 973-986). Although high-sensitivity immunoassays can detect elevated troponin levels, because antibodies have batch-to-batch variations and target different epitopes, these assays often yield inconsistent results and contribute to false observations of elevated cTnI levels in non-acute myocardial infarction patients (Herman et al., 2017, American Journal of Clinical Pathology, 148 (4): 281-295; and Hyytiä et al., 2015, Clinical Biochemistry, 48(4): 313-317). Current patient risk-stratification and heterophile antibodies also lead to high false positive rates and increased costs (Zaidi et al., 2010, BMJ Case Rep., 2010:bcr1120092477).
Importantly, in reviewing troponin testing (qualitative point-of-care and quantitative cTnI measurements) at 14 hospitals, only 3.5% of 92,445 elevated troponin values (>0.09 ng/ml) were associated with acute myocardial infarction (AMI) diagnosis. While current cTnI assays have high negative predictive value (>99%) leading to few cases of “false-negative” AMI diagnosis, the positive predictive value (28.8%) is low (Wilson et al., 2017, J. Hosp Med, 5: 329-331). The continued use of current cTnI antibody-based immunoassays for AMI leads to increased cardiology consultation and medical testing, contributing significantly to the $200 billion spent in cardiovascular related healthcare costs per year.
Moreover, because cTnI typically exists as a myriad of proteoforms in low-abundance which arise from genetic variations, alternative splicing, and post-translational modifications (PTMs) for a single gene product, the unambiguous identification and characterization of cTnI proteoforms remains a significant challenge for the antibody-based approach. However, proteoform-resolved information is essential to accurate diagnosis of AMI. Furthermore, because dysregulation of cTnI proteoforms occurs in AMI and other cardiomyopathies, an assay that unambiguously characterizes cTnI proteoforms in blood is essential for accurate diagnosis of AMI (Soetkamp et al., 2017, Review of Proteomics 14: 973-986), and also for risk stratification, and outcome assessment for patients with acute coronary syndrome (ACS) and non-ACS myocardial injury. Accordingly, there is an urgent need to develop an accurate and comprehensive assay that combines cTnI enrichment from blood with sensitive mass spectrometry (MS)-based validation to detect and comprehensively characterize all proteoforms of cTnI.
Top-down MS-based proteomics, which analyzes intact proteins, is arguably the most powerful method to comprehensively and accurately characterize proteoforms, including those of cTnI (Chen et al., 2018, Analytical Chemistry 90(1): 110-127; Siuti et al., 2007, Nature Methods 4: 817-821; and Kelleher et al., 2014, Expert Review of Proteomics 11(6): 649-651). However, MS-based proteomics is limited by the large dynamic range and the complexity of the human blood proteome, and often requires additional front-end enrichment strategies (Anderson et al., 2002, History, Character, and Diagnostic 1(11): 845-867). Because cTnI is present at low concentrations in human serum, proteomics analysis without prior enrichment of cTnI would only result in the detection of the most abundant blood proteins. Thus, the development of front-end MS-compatible strategies that can enrich cTnI prior to MS-analysis is critical.
The present invention provides mass spectrometry (MS) compatible nanomaterials for the selective capture and enrichment of biomolecules, preferably proteins, including, but not limited to, cardiac proteins. Nanoparticles are functionalized with probe molecules that can specifically bind to a desired biomolecule (or a desired class of biomolecule) and allow for accurate MS-analysis and characterization of the biomolecule(s). In the case where the desired biomolecules are proteins, this analysis and characterization can include top-down MS-based proteomic analysis of intact, unfragmented or undigested proteins as well analysis of different proteoforms of the proteins.
In an embodiment, the present invention provides a composition comprising a nanoparticle and one or more probe molecules attached to the nanoparticle, wherein the one or more probe molecules are able to preferentially bind to a selected biomolecule (or a class of biomolecules). The nanoparticle is preferably a superparamagnetic nanoparticle, including but not limited to nanoparticles comprising magnetic ferrites. In an embodiment, the nanoparticle comprises Fe3O4, Fe2O4, CoFe2O4, ZnFe2O4, NiFe2O4, MnFe2O4, and combinations thereof. In an embodiment, the nanoparticle comprises iron oxide (Fe3O4) or cobalt ferrite (CoFe2O4). In an embodiment, nanoparticle comprises iron oxide (Fe3O4).
Preferably, the surface of the nanoparticle is functionalized with one or more organosilane coupling molecules, and the one or more probe molecules are attached to the nanoparticle through the one or more coupling molecules. In a further embodiment, the one or more coupling molecules comprise amine based organosilane coupling molecules, monomers, amine based organosilane monomers, and combinations thereof. In an embodiment, the one or more coupling molecules comprise N-(3(triethoxysilyl) propyl)buta-2,3-dienamide (BAPTES), N-(3(trimethoxysilyl) propyl)buta-2,3-dienamide (BAPTMS), N-(3(triethoxysilyl) propyl)-3-butynamide, N-(3(trimethoxysilyl) propyl)-3-butynamide, or combinations thereof.
In an embodiment of the present invention, the surface of a nanoparticles is functionalized with one or more probe molecules, including but not limited to polypeptides that can preferentially bind to a desired protein. These polypeptides can include antibodies and fragments of antibodies; however, the present invention is not limited to the use of antibodies to bind to the desired protein. Preferably, the nanoparticles are functionalized with probe molecules having a high affinity and selectivity for a desired protein, especially a desired proteoform. Useful proteoforms include, but are not limited to, phosphorylated proteoforms, unphosphorylated proteoforms, degraded proteoforms, glycosylated proteoforms, oxidized proteoforms, acetylated proteoforms, post-translational modified proteoforms, and combinations thereof.
In an embodiment, the nanoparticles are functionalized with probe molecules having a high affinity and selectivity for a cardiac protein, including but not limited to cardiac troponin I (cTnI) or cardiac troponin T (cTnT) within the human cardiac troponin complex, as well as proteoforms of cTnI and cTnT. Optionally, the nanoparticle utilizes the modular attachment of a cysteine (thiol)-terminated peptide with high affinity and selectivity for cTnI, allowing for the capture, enrichment, and MS-analysis and characterization of cTnI proteoforms from human heart tissue lysates and human blood and/or serum samples. This invention further provides a comprehensive and accurate cTnI assay for detection of cTnI from patient blood and/or serum samples. Such assays are useful for the accurate diagnosis of acute coronary syndrome (ACS) and chronic diseases, including but not limited to acute myocardial infarction (AMI). Such assays are also useful for risk stratification, and outcome assessment for patients with ACS and non-ACS myocardial injury.
In an embodiment, the surface of the nanoparticles is functionalized with polypeptides having a binding affinity (Kd) of at least 200 pM to the selected protein, preferably a binding affinity (Kd) of at least 270 pM to the selected protein. In an embodiment, the one or more polypeptides comprise an amino acid sequence having at least 75% sequence identity to HWQIAYNEHQWQ (SEQ ID NO: 1), preferably at least 80% sequence identity to HWQIAYNEHQWQ, preferably at least 90% sequence identity to HWQIAYNEHQWQ. In an embodiment, the one or more polypeptides comprise the amino acid sequence HWQIAYNEHQWQ. In an embodiment, the one or more polypeptides comprise an amino acid sequence having at least seven contiguous amino acids of SEQ ID NO: 1, preferably at least eight contiguous amino acids, preferably at least nine contiguous amino acids, preferably at least ten contiguous amino acids, and preferably at least eleven contiguous amino acids. Short (<20 amino acids) and medium sized polypeptides (between 20 and 100 amino acids) are pH and temperature stable, and are easier to specifically functionalize compared to typical antibodies. For example, addition of a cysteine-residue to the C-terminus of such a peptide (i.e., HWQIAYNEHQWQ-Cys—SEQ ID NO:2) allows for novel, chemoselective nanoparticle surface coupling chemistry. Optionally, the functionalization happens through allenic amide-based organosilane monomers that are highly specific and reactive towards thiol-containing molecules of peptides in the presence of other biologically relevant nucleophiles, such as hydroxyls, amines, and carboxylates.
In an embodiment, the surface of the nanoparticles is functionalized with a small molecule affinity reagent that is modified with a cysteine-thiol linker. Such small molecule affinity reagents include, but are not limited to, kinase inhibitors, GPCR agonists and GPCR antagonists for kinases or G-protein coupled receptors and ACE2 receptors. Preferably, the small molecule affinity reagent has a binding affinity (Kd) of at least 200 pM to the selected protein, or more preferably a binding affinity (Kd) of at least 270 pM to the selected protein.
As used herein, the nanoparticles have a diameter of 100 nm or less, preferably 40 nm or less, or 10 nm or less.
In an embodiment, the present invention provides a method of making a functionalized nanoparticle comprising a superparamagnetic nanoparticle and one or more probe molecules attached to the nanoparticle, where the method comprising the steps of: a) silanizing at least a portion of a surface a superparamagnetic nanoparticle with one or more amine-based organosilane monomers, wherein said one or more monomers comprise a functional group having high chemoselectivity towards thiol-containing molecules; and b) reacting the silanized nanoparticle with a probe molecule or probe molecule precursor having a cysteine amino acid residue or a terminal thiol functional group. The probe molecule or probe molecule precursor is optionally a polypeptide having a terminal cysteine residue, and the functional group is optionally an allene functional group. In an embodiment, the one or more monomers preferably comprise N-(3(triethoxysilyl)propyl)buta-2,3-dienamide (BAPTES) and the probe molecule precursor comprises the amino sequence HWQIAYNEHQWQ-Cys (SEQ ID NO:2).
In an embodiment, the present invention provides a method for analyzing a cardiac protein in a sample, said method comprising the steps of:
a) adding functionalized nanoparticles to the sample containing the cardiac protein, wherein the functionalized nanoparticles comprise a superparamagnetic nanoparticle and one or more probe molecules attached to the superparamagnetic nanoparticle, wherein the one or more probe molecules are able to preferentially bind to the cardiac protein, thereby generating protein bound nanoparticles;
The method optionally further comprises purifying the enriched fraction prior the MS analysis, such as by method including, but not limited to, various modes of liquid chromatography (LC) methods.
Preferably, the one or more probe molecules are able to preferentially bind to a cardiac protein having a molecular weight of 80 kDa or less, preferably 60 kDa or less, or 30 kDa or less, including but not limited to cardiac troponin I (cTnI) or cardiac troponin T (cTnT) as well as various proteoforms of cTnI and cTnT. The sample comprises blood, serum, plasma, tissue, or combinations thereof, taken from a subject (preferably a human subject).
In an embodiment, the sample is taken from a patient and a diagnosis of a cardiac disease (or cardiac diseases) is performed based on presence of the cardiac protein in the sample. The cardiac disease comprises ACS and non-ACS chronic diseases, including but not limited to AMI. Optionally, the cardiac protein is a selected proteoform of a cardiac protein, preferably a proteoform of cTnI, and the method comprises binding one or more proteoforms of the cardiac protein to the functionalized nanoparticles, magnetically isolating the protein bound nanoparticles, eluting the one or more proteoforms, and ionizing and performing MS analysis on the one or more proteoforms. The diagnosis of the cardiac disease is based on the relative amount of one proteoform to another proteoform from the sample, or to the amount of the proteoform compared to a control sample of a healthy population. Useful proteoforms for the diagnosis of cardiac diseases include, but are not limited to, phosphorylated proteoforms, unphosphorylated proteoforms, degraded proteoforms, glycosylated proteoforms, oxidized proteoforms, acetylated proteoforms, post-translational modified proteoforms, and combinations thereof.
In a further embodiment, the method further comprises taking a first sample from the patient at a first time period, taking one or more subsequent samples from the patient at one or more later time periods, and comparing the relative amounts of the one or more proteoforms from the first sample and the one or more subsequent samples.
Cardiovascular diseases are the leading causes of death globally (Benjamin et al., 2018, Circulation, 137: e67-e492), account for approximately one out of every four deaths in the United States, and further place an enormous financial burden on the healthcare system. Early and accurate diagnosis of heart failure enables successful patient outcomes and reduces the need for excessive and costly testing.
Cardiac troponin I (cTnI) is an important protein in cardiomyocytes that regulates cardiac muscle contraction (Lemos et al., 2013, JAMA, 309:2262). cTnI is also clinically recognized as a sensitive and specific ‘gold-standard’ protein biomarker for acute coronary syndrome because it has an amino acid sequence specific to the cardiac tissue and is released into the bloodstream following cardiac injury (Missov et al., 1997, Circulation, 96:2953-2958) with an increased circulating cTnI concentration correlated to the onset of cardiac damage (Thygesen et al., 2018, Journal of the American College of Cardiology, 25285; Westermann et al., 2017, Nature Reviews Cardiology, 14: 472; Antman et al., 1996, New England Journal of Medicine 335: 1342-1349). Increased cTnI levels in the bloodstream are associated with myocardial injuries such as AMI and ischemia. To date, antibody-based assays testing for elevated levels of cTnI in the bloodstream are the primary tests used to diagnose AMI.
However, high-sensitivity immunoassays used to detect elevated cTnI levels often yield inconsistent results and contribute to false observations of elevated cTnI levels in non-AMI patients. While current cTnI assays have a high negative predictive value (>99%) leading to very few cases of “false-negative” AMI diagnosis, the positive predictive value (28.8%) is low. As a result, current cTnI antibody-based immunoassays contribute to increased cardiology consultation and medical testing. Thus, an improved assay for accurately detecting cTnI is of interest for accurate diagnosis, risk stratification, and outcome assessment for patients with ACS and non-ACS myocardial injury.
Additionally, circulating cTnI in the blood exists in low abundance and in myriad proteoforms (Smith et al., 2013, Nature Methods 10: 186), such as phosphorylated, truncated, acetylated, and oxidized proteoforms, that are known to reflect pathophysiological processes (Bates et al., 2010, Clinical Chemistry, 56: 952; Madsen Lene et al., 2006, Circulation Research, 99: 1141-1147; and Soetkamp et al., 2017, Expert Review of Proteomics 14: 973-986). Current antibody-based enzyme-linked immunosorbent assays (ELISAs) for cTnI are incapable of distinguishing these modified cTnI proteoforms, and elevated cTnI concentrations indirectly quantified by ELISA are not indicative of the pathology underlying cardiac myocyte injury (Soetkamp et al., 2017, Expert Review of Proteomics 14: 973-986; and Labugger et al., 2000, Circulation, 102: 1221-1226). Moreover, the intrinsic limitations of immunoassays such as batch-to-batch variation, heterophilic antibody interference, and lack of standardization in the use of antibodies targeting different epitopes yield inconsistent results across laboratories and a high false discovery rate (Twerenbold et al., 2017, Journal of the American College of Cardiology 70: 996-1012; and Tighe et al., 2015, PROTEOMICS— Clinical Applications 9: 406-422). This further contributes to unnecessary and costly health care expenditures.
To address these problems, the examples below provide a proteoform-resolved comprehensive cTnI assay enabled by an integrated nanoproteomics strategy for the specific capture and enrichment of cTnI using functionalized nanoparticles (NPs) followed by top-down mass spectrometry (MS) analysis of various cTnI proteoforms. This nanoproteomics strategy is antibody-free, simple, scalable, and highly reproducible with negligible batch-to-batch variations. The examples demonstrate specific enrichment of cTnI from serum while simultaneously depleting highly abundant serum proteins and importantly, direct detection and quantification of cTnI proteoforms from serum with a limit of detection as low as 0.75 ng/ml. Such proteoform-resolved cTnI assay reveals previously unachievable in-depth molecular details of cTnI as proteoform-specific biomarkers for diagnosis of cardiac diseases with high accuracy.
The nanoparticle strategy described herein for cTnI enrichment is superior to existing method based on antibody-conjugated microparticles for the following reasons: (1) nanoparticles (NPs) are comparable in size to typical proteins to enhance protein capture and enrichment and can penetrate better in complex protein mixtures, leading to a higher interaction rate; (2) this platform enables modular functionalization of the NP surface by accessing the unique reactivity of the allenecarboxamide motif for specific thiol-conjugation of biomolecular recognition molecules and various other small molecules; (3) as biomolecular recognition elements, peptides are synthesized easily and reproducibly at low cost, are pH and temperature stable, and have considerably longer shelf-life compared to antibody-based reagents; and (4) these functionalized NP reagents can be synthesized in large scale with good quality control, which could significantly lower the cost and further minimize the batch-to-batch variation common to antibody production.
Recent studies have convincingly shown that cTnI is heavily modified and its proteoforms arising from various post-translational modifications (PTMs) can provide new insights to the molecular mechanisms underlying various cardiovascular diseases (Soetkamp et al., 2017, Expert Review of Proteomics 14: 973-986; and Labugger et al., 2000, Circulation, 102: 1221-1226). Therefore, a comprehensive and accurate proteoform-resolved cTnI assay is urgently needed. Top-down MS proteomics is ideally suited for this major challenge because it analyzes intact proteins and is the most powerful method to comprehensively characterize proteoforms and decipher PTMs (Chen et al., 2018, Analytical Chemistry, 90: 110-127; Siuti et al., 2007, Nature Methods, 4: 817-821). However, the high dynamic range of the blood proteome (1012) makes detection of low-abundance proteins (such as cTnI) extremely difficult, especially directly from blood serum samples in the presence of many highly abundant proteins such as human serum albumin (HSA) (Anderson et al., 2002, History, Character, and Diagnostic Prospects 1: 845-867). Therefore, protein enrichment is required prior to MS analysis (Anderson et al., 2002, History, Character, and Diagnostic Prospects 1: 845-867; Xie et al., 2009, Expert review of proteomics 6, 573-583; and Smith et al., 2017 Circulation 135: 1651-1664). The following nanoproteomics strategy was therefore developed using surface functionalized magnetic NPs that can capture and enrich cTnI directly from human serum, followed by top-down MS to enable a comprehensive proteoform-resolved cTnI assay.
Firstly, a surface-functionalized superparamagnetic iron oxide (magnetite, Fe3O4) NPs (Park et al., 2004, Nature Materials, 3: 891-895) was designed for the capture and specific enrichment of cTnI from complex mixtures (
Instead of using antibodies to target cTnI, a short, linear peptide was chosen which was evolved for high cTnI affinity by phage display and in silico evolution (Xiao et al., 2018, ACS Sensors 3: 1024-1031). The use of peptides offers attractive properties for protein enrichment in comparison with antibodies, such as improved chemical stability to changes in pH and reducing environments, thermal stability, scalability using solid-phase peptide synthesis, and batch-to-batch reproducibility. This specific peptide (HWQIAYNEHQWQ—SEQ ID NO:1) not only exhibits an impressive binding affinity (Kd) of 270 pM comparable to that of antibodies (Xiao et al., 2018, ACS Sensors 3: 1024-1031), but also targets a central portion of cTnI (amino acid residues 114-144) that is less susceptible to proteolysis and postulated to be an optimal targeting epitope to detect all forms of cTnI present in blood circulation (Bates et al., 2010, Clinical Chemistry 56: 952; Katrukha et al., 1998, Clinical Chemistry 44: 2433; and Apple et al., 2012, Clinical Chemistry 58: 54).
To evaluate the peptide functionalization approach, the reaction of BAPTES with a C-terminal Cys-modified derivative of the high affinity peptide (HWQIAYNEHQWQC— SEQ ID NO:2) was first analyzed using high-resolution tandem MS (MS/MS). The peptide-Cys' reaction with BAPTES occurred exclusively even in the presence of other biologically relevant nucleophiles, such as hydroxyls, amines, and carboxylates (
Following confirmation of such allenecarboxamide coupling chemistry, the BAPTES silanized NPs (NP-BAPTES) were then functionalized with the high affinity cTnI binding-peptide onto the NP surface, which is referred to as NP-Pep hereafter (
After peptide coupling, the relative intensities of the characteristic allene peaks in the FTIR spectra were reduced, indicating the successful consumption of the allene groups upon peptide conjugation. Thermogravimetric analysis (TGA) of the NP-BAPTES revealed a 28% weight loss, accounting for a thin silane coating on the surface functionalized NPs (
All errors indicate standard deviations from measurements on three independent batches of particles.
The cTnI enrichment performance of the NP-Pep were then evaluated using sarcomeric protein extracts prepared from human heart tissues (Table 2). SDS-PAGE was used to visualize the sarcomeric extracts, which was found to contain approximately 0.3% cTnI by ELISA, to confirm the reproduciblity of our protein extraction procedure (
It was further investigated whether high affinity cTnI-binding peptide functionalization onto the NPs was a critical factor for the high specificity enrichment by using the following NP controls: (1) NP-BAPTES as ‘NP-Ctrl’ with no peptide; (2) NP-BAPTES functionalized with peptide at an acidic pH to inhibit formation of the covalent Cys-thiol conjugate; (3) NP-BAPTES functionalized with a negative-control peptide containing alanine substitutions to reduce cTnI-binding affinity (
The cTnI enrichment performance of the NP-Pep were evaluated by top-down LC/MS analysis of the initial protein loading mixture, the resulting flow-through, and the final elution mixtures after cTnI enrichment by the NP-Pep, which allowed a bird's eye view of all cTnI proteoforms present for direct quantification of the ratios between modified cTnI proteoforms normalized to total cTnI. Importantly, the NP-Pep preserved all endogenous cTnI proteoform distributions and faithfully retained the endogenous cTnI PTM ratios at every step of the enrichment process with no artifactual modifications (
To illustrate the unique advantages of the NP-Pep system for cTnI enrichment compared to existing affinity purification systems, the cTnI enrichment performance of the NPs were then compared against a conventional agarose-based solid support functionalized with the same high affinity cTnI-binding peptide (Agarose-Pep) as well as with an anti-cTnI monoclonal antibody (M46;Agarose-mAb) that possesses a similar cTnI-binding epitope (amino acids 130 to 145) to that of the high affinity peptide (
Furthermore, rapid top-down LC/MS coupling reversed phase liquid chromatography (RPLC) to high-resolution MS revealed the molecular nature of the endogenous cTnI proteoforms found in the loading mixture (L), flow through (F) and elution (E) for each cardiac sample (lower panels of
Although top-down MS methods have been developed for the comprehensive analysis of various cTnI proteoforms simultaneously after they are extracted from tissues (Dong et al., 2012, Journal of Biological Chemistry 287: 848-857; Zhang et al., 2011, Journal of Proteome Research 10: 4054-4065; and Zabrouskov et al., 2008, Mol Cell Proteomics 7: 1838-1849), MS detection of low abundance cTnI in the blood remains an unsolved challenge due to the extreme complexity and dynamic range (˜1012) of the blood proteome and the presence of highly abundant blood proteins (˜90% of total blood mass) such as human serum albumin (HSA) (Anderson et al., 2002, History, Character, and Diagnostic Prospects 1: 845-867; and Rifai et al., 2006, Nature Biotechnology 24: 971-983). To this end, cTnI enrichment was performed from human serum spiked-in with the cTnI extracted from the same donor, DCM, and post-mortem heart samples as in
Even though cTnI was undetectable in the original serum mixture due to the low abundance of cTnI compared to the higher abundance blood proteins, the NP-Pep elution fraction showed nearly complete depletion of HSA and significant enrichment of cTnI relative to the loading mixture (
These results illustrate the remarkable benefits of the functionalized NPs compared to conventional antibody-based approaches: (1) the NPs are capable of effective cTnI enrichment across all tested serum samples and cardiac pathologies; (2) the NPs retain all endogenous cTnI proteoforms from serum without introducing artifactual modification; (3) the NPs show reproducible and highly significant depletion of HSA after NP-Pep enrichment (
After determining the absolute cTnI amount using an ELISA assay (
After successfully enriching cTnI from human serum, this nanoproteomic strategy was applied to six different human serum samples each consisting of unique cTnI references obtained from different donor, diseased, or post-mortem heart tissues to mimic the enormous diversity of circulating cTnI proteoforms that could be found in blood for demonstrating the comprehensive analysis of cTnI from human serum (
In summary, this example demonstrates the first comprehensive proteoform-resolved cTnI assay enabled by nanotechnology and top-down MS. Carefully designed surface functionalized NPs can directly capture and enrich cTnI from blood with high specificity and reproducibility, while simultaneously depleting high-abundance blood proteins such as HSA. The proteoforms of enriched cTnI could be detected and quantified by a rapid LC/MS-based top-down proteomics with high sensitivity (LOD 0.75 ng/mL). Furthermore, all cTnI proteoforms could be completely recovered after a highly specific and effective enrichment from serum by NP-Pep and the cTnI proteoform signature resolved by LC-MS can be directly linked to the phenotypes. This nanoproteomics cTnI assay could be further developed into a clinical diagnostic assay after testing in a large human cohort by specifically enriching cTnI from patient blood samples and comprehensively detecting all cTnI proteoforms to establish a robust relationship between cTnI proteoforms and disease etiology. It is envisioned that this nanoproteomics-enabled cTnI assay will be the next generation comprehensive assay to provide previously unachievable high-resolution proteoform-resolved molecular signature of cTnI for accurate diagnosis, prognosis, and risk stratification of patients with ACS and non-ACS chronic diseases toward precision medicine.
ELISA-based cTnI enrichment efficiency quantification of NP-Pep and Agarose-mAb. ELISA-based colorimetric quantification of cTnI standards (left dashed box) and enrichment samples (right dashed box) were performed (
Top-down LC-MS/MS characterization of cTnI arising from the various biological samples after enrichment. Representative CID fragment ions obtained from cTnI arising from donor heart (y7613+, y20930+, b8612+, and b16621+), diseased heart (y7612+, y427+, b315+, and b8612+), and post-mortem heart (y768+, y619+, b8614+, and b315+) sources are shown in
Preservation of cTnI PTM profiles by NP-Pep from serum reflecting different cardiac pathophysiologies.
Materials and Reagents. All chemicals and reagents were purchased from MilliporeSigma (St. Louis, Mo., USA) and used as received without further purification unless otherwise noted. Sodium oleate (97%), was purchased from Tokyo Chemical Industry (TCI) America (Portland, Oreg., USA). The high affinity cTnI peptide (95%) with C-terminal Cysteine residue (HWQIAYNEHQWQC—SEQ ID NO: 2) and the nonspecific peptide (95%) with C-terminal Cysteine residue (HWNMAANEHMQWC—SEQ ID NO: 3) were purchased from GenScript USA Inc. (3-aminopropyl)triethoxysilane (APTES) was purchased from Gelest (Morrisville, Pa., USA). Human male AB serum (H4522) was purchased from Millipore Sigma (St. Louis, Mo., USA). Human cardiac troponin I-C monoclonal antibody (M46, cat. #sc-52277) and phosphatase inhibitor cocktail A (cat. #sc-45044) were purchased from Santa Cruz Biotechnology, Inc. N-hydroxysuccinimide (NHS) activated agarose slurry (cat. #26200) and Halt™ Protease inhibitor cocktail were purchased from ThermoFisher Scientific (Rockford, Ill., USA). Human cardiac troponin I ELISA kit (AccuBind® ELISA, cat. #3825-300) was purchased from Monobind Inc. (Lake Forest, Calif., USA). Extraction solutions were made in nanopure deionized water (H2O) from Milli-Q® water (MilliporeSigma). Bradford protein assay reagent was purchased from Bio-Rad (Hercules, Calif., USA). 12.5% gel (10 comb or 15 comb well, 10.0 cm×10.0 cm, 1.0 mm thick) for SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was home-made. DynaMag™-2 Magnet and Thermo Scientific™ Cimarec+™ stirring hotplate were purchased from ThermoFisher Scientific (Rockford, Ill., USA). Amicon, 0.5 mL cellulose centrifugal filters with a molecular-weight cutoff (MWCO) of 10 kDa was purchased from MilliporeSigma.
Synthesis of But-3-nyoic acid. 3-butynoic acid was prepared from the oxidation of 3-butyn-1-ol following the reported procedure (Abbas et al., 2014, Angewandte Chemie International Edition, 53(29): 7491-7494). 135 mL water was added to a 500 mL single neck RBF fitted with magnetic stirrer bar. 65% HNO3 (0.51 mL, 5 mol %, 7.5 mmol), Na2Cr2O7 (0.45 g, 1 mol %, 1.5 mmol) and NalO4 (70.60 g, 2.2 eq., 330 mmol) were subsequently added to the RBF and the mixture was stirred vigorously on an ice bath for 15 min. 11.3 mL of 3-butyn-1-ol (1 eq., 150 mmol) dissolved in 135 mL of chilled water was added to this mixture slowly and the reaction mixture was stirred overnight. After this time, the product was extracted in diethyl ether (100 mL×6). All the fractions were combined and dried over anhydrous magnesium sulfate. Solvent was evaporated on a rotary evaporator to give an orange/yellowish viscous liquid. Subsequent addition of dichloromethane and removal of solvent under vacuum 4-5 times yielded 6.56 g of an off white/yellowish solid (52% yield). 1H NMR (500 MHz, Chloroform-d) δ 3.38 (d, 2H, J=2.7 Hz), 2.25 (t, 1H, J=2.7 Hz); 13C NMR (125 MHz, Chloroform-d) δ 173.8, 74.8, 72.4, 25.6.
Synthesis of N-(3-(triethoxysilyl)propyl)buta-2,3-dienamide (BAPTES). But-3-nyoic acid (2.10 g, 25.0 mmol, 1 eq.), 2-chloro-1-methylpyridinium iodide (6.39 g, 25.0 mmol, 1 eq.), and CH2Cl2 (250.0 mL, 0.10 M) were added to an oven dried 500 mL three-neck flask fitted with a reflux condenser. The solution was heated to reflux under N2. In a separate flask, (3-aminopropyl)thiethoxysilane (5.85 mL, 25.0 mmol, 1 eq.) and N,N-diisopropylethylamine (8.71 mL, 50.0 mmol, 1 eq.) were diluted with 125.0 mL CH2Cl2, and added to the refluxing three-neck flask by syringe. The reaction mixture was allowed to reflux for 1 hour, and then N,N-Diisopropylethylamine (4.36 mL, 25.0 mmol, 1 eq.) was additionally added to fully isomerize the propargylic isomer to N-(3-(triethoxysilyl)propyl)buta-2,3-dienamide. The reaction was allowed to reflux overnight, and the product was concentrated by rotary evaporation. The crude product was suspended in ethyl acetate, centrifuged at 5,000 rpm for 5 min, and the supernatant was concentrated by rotary evaporation. The product was purified with flash column chromatography using a gradient of 50:50 ethyl acetate: hexane to 100:0 ethyl acetate: hexane to obtain a clear, orange oil (67% yield). 1H NMR (500 MHz, Chloroform-d) δ 5.99 (s, 1H), 5.62 (t, J=6.6 Hz, 1H), 5.20 (d, J=6.7 Hz, 2H), 3.83 (q, J=7.0 Hz, 6H), 3.31 (q, J=6.7 Hz, 2H), 1.66 (m, 2H), 1.24 (t, J=7.0 Hz, 9H), 0.66 (m, 2H).
Synthesis of iron-oleate precursor. Iron oleate was synthesized using a previously established method (Park et al., 2004, Nature Materials, 3(12), 891-895). In a typical synthesis, iron (III) chloride hexahydrate (10.8 g, 40 mmol) was first dissolved in a mixture of 80 mL ethanol and 60 mL nanopure water in a three-neck round bottom flask (500 mL) containing a Teflon-coated egg-shaped (1¼″×⅝″) magnetic stir bar. Sodium oleate (36.5 g, 120 mmol) was then quickly added to the iron chloride solution along with 140 mL n-hexane. The resulting solution was then allowed to stir until the sodium oleate was completely dissolved. Afterwards, the reaction solution was heated to 70° C. for a 4 h reflux under a N2 blanket. Upon completion, the reaction solution was cooled to room temperature, and the upper organic layer containing the iron oleate was washed three times with 30 mL nanopure water in a 250 mL separatory funnel. After washing, excess hexane was then removed by rotary evaporation. Finally, the resulting iron oleate was transferred into a 100 mL round bottom flask, connected to a Schlenk line, and placed under vacuum overnight. For later storage, the iron oleate was well sealed in a glass vial and placed in a desiccator.
Synthesis of 8 nm magnetite (Fe3O4) nanocrystals. Iron oleate (10 mmol, 9.0 g) and oleic acid (5.5 mmol, 1.56 g) were added into a three-neck round bottom flask (250 mL) with a solvent mixture of 1-octadecene:1-tetradecene (40 g: 10 g). The mixture was stirred and degassed on a Schlenk line at 110° C. for 3 h. The mixture was then placed under nitrogen flow and the reaction solution was heated to 300° C. at a heating rate of 3.3° C./min using a temperature controller. Reaction time was counted starting from when 300° C. was reached. After 1 h, the reaction solution was quickly cooled to room temperature by blowing air across the reaction flask. The resulting iron-oxide nanocrystals were precipitated using ethanol, isolated via centrifugation (5,000 rpm, 20 min), and then washed with three precipitation/redispersion cycles (5,000 rpm, 20 min) using ethanol. The resulting nanocrystals were then dried under vacuum, weighed, and redispersed in n-hexane at a concentration of 20 mg/mL for further use.
Synthesis of Fe3O4-BAPTES NPs (NP-BAPTES). In a typical optimized large-scale synthesis of silane functionalized NPs, Fe3O4 NPs (6 mL from a 20 mg/mL stock) were added to anhydrous n-hexane (300 mL) in a 500 mL round bottom flask equipped with a Teflon-coated egg-shaped magnetic stir bar (1¼″×5″) to achieve a total NP concentration of 0.4 mg/mL. After the reaction mixture was heated to 60° C. with stirring (900 rpm), BAPTES (1.65 mL) was added dropwise to the flask for a 0.55% (v/v) total concentration of trialkoxysilane reagent, followed by dropwise addition of a small amount of acetic acid (30 μL) for an acidic catalyst concentration of 0.01% (v/v). After reaction under stirring for 24 h, the precipitate was collected and washed one time with n-hexane, one time with n-hexane/acetonitrile (v/v, 4:1), and one more time with n-hexane via centrifugation (5,000 rpm, 10 min) to remove excess silane molecules and surfactants. The NPs were then dried under vacuum for later use.
Synthesis of Fe3O4-BAPTES-Peptide NPs (NP-Pep). 10 mg of NP-BAPTES were added to a 4-dram vial and dispersed in 2 mL of acetonitrile. 10 mg of cTnI-binding peptide (HWQIAYNEHQWQC—SEQ ID NO: 2) were added to a separate 4-dram vial and dissolved in 8 mL of nanopure water. The pH of the peptide solution was adjusted to pH 8.0 by the addition of 75 μL of 1.0 M ammonium carbonate buffer pH 9.0 during simultaneous water bath sonication. The pH-adjusted peptide solution was added into the 4-dram vial containing the NP dispersion under water bath sonication. The NP reaction mixture was allowed react under sonication for 1 h and later collected into Eppendorf tubes for washing. The peptide-functionalized NPs were washed three times with water via centrifugation (15,000 rcf, 5 min) and subsequently isolated magnetically with a DynaMag to remove unreacted peptide. The resulting peptide functionalized NPs were redispersed in water at a concentration of 5 mg/mL.
Material characterization. Transmission electron microscopy (TEM) samples were prepared by pipetting a 10 μL drop of as-synthesized NPs at a concentration of 0.125 mg/mL onto a copper TEM grid with lacey carbon film. TEM was conducted on a FEI T12 microscope operated at 120 kV, equipped with a Gatan CCD image system with digital micrograph software program. Transmission Fourier transform infrared (FTIR) spectroscopy measurements were recorded on a Bruker Equinox 55 FT-IR spectrometer in the range of 4,000 cm−1 to 400 cm−1 at 2 cm−1 resolution on NPs in a potassium bromide (KBr) pellet, at a sample mass loading of 0.33 wt. %. Thermogravimetric analysis (TGA) was carried out using a TA Instruments Q500 thermal analysis system under a N2 atmosphere and at a constant heating rate of 10° C./min from 100° C. to 600° C. All samples were first heated to 100° C. and held at that temperature for 3 min to remove adsorbed water.
Small molecule analysis by FTICR-MS. All small molecule samples (<3 kDa) were diluted 500-fold in 50:50:0.1 (acetonitrile:water:formic acid) MS-grade solvent for positive electrospray ionization mode analysis. Samples were analyzed by direct infusion using a TriVersa Nanomate system (Advion BioSciences, Ithaca, N.Y.) coupled to a solariX XR 12-Tesla Fourier Transform Ion Cyclotron Resonance Mass spectrometer (FTICR-MS, Bruker Daltonics). For the nano-electrospray ionization source (TriVersa Nanomate), the desolvating gas pressure was set at 0.5 PSI and the voltage was set to 1.2 to 1.6 kV versus the inlet of the mass spectrometer. Mass spectra were acquired with an acquisition size of 1M, in the mass range between 150 and 2000 m/z (with a resolution of 270,000 at 400 m/z), and 50 scans were accumulated for each sample. Ions were accumulated in the collision cell for 0.05 s, and a time of flight of 0.500 ms was used prior to their transfer to the ICR cell. For collisionally activated dissociation MS/MS experiments, the collision energy was varied from 10 to 20V. Tandem mass spectra were output from the DataAnalysis software and analyzed using MASH Suite Pro software. All the program-processed data were manually validated. The methods described here correspond to the data presented in
Sarcomeric protein extraction from human cardiac tissue samples. All protein extraction procedures were performed in a cold room (4° C.) using freshly prepared buffers. All human cardiac tissue was obtained from the University of Wisconsin Hospital and Clinic. The procedure for the collection and de-identification of human cardiac tissue was approved by the Institutional Review Board of the University of Wisconsin-Madison. 500 mg of tissue was homogenized in wash buffer (5 mM NaH2PO4, 5 mM Na2HPO4, 5 mM MgCl2, 0.5 mM EGTA, 0.1 M NaCl, 1% Triton X-100, 5 mM DTT, 1 mM PMSF, 1× HALT protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail A, pH 7.4) using a Polytron electric homogenizer (Model PRO200; PRO Scientific, Oxford, Conn., USA) on ice. The resulting homogenate was centrifuged at 10,000 g (Avanti J-25i; Beckman Coulter, Fullerton, Calif., USA) for 10 min at 4° C. After centrifugation, the supernatant was removed, and the pellet was washed once more with wash buffer. The wash supernatant was removed, and the pellet was re-suspended in protein extraction buffer (0.7 M LiCl, 25 mM Tris, 5 mM EGTA, 0.1 mM CaCl2, 5 mM DTT, 1 mM PMSF, 1× HALT protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail A, pH 7.5), and the suspension was agitated on a nutating mixer (Thermo Scientific, Boston, Mass., USA) at 4° C. for 45 min. The sarcomeric protein extract was centrifuged at 16,000 g (Centrifuge 5415R, Eppendorf, Hamburg, Germany) for 10 min at 4° C. and the resulting supernatant was again centrifuged at 21,000 g for 30 min to remove all tissue debris. The concentration of the tissue lysate was determined by Bradford protein assay. Samples were stored at −80° C. for later study. The buffers and reagent preparation for the methods described here correspond to the data presented in Table 2.
cTnI enrichment using NP-Pep from human cardiac sarcomere extracts. NP-Pep (5 mg) was resuspended in a 2 mL Eppendorf Protein Lo-Bind tube with equilibration buffer (50 mM Tris, pH 7.5, 150 mM LiCl). The NPs were then centrifuged at 15,000 rcf for 2 min at 4° C., isolated from the solution using the DynaMag, and the supernatant was removed. Equilibration buffer was then added to the NPs, the mixture was sonicated and vortexed to prepare for protein loading. Protein loading mixture (L), from tissue extract was diluted to a final volume of 1 mL with a buffered solution (50 mM Tris, pH 7.4, 150 mM LiCl) to a total protein loading of 0.3 mg/mL and was added to the NP-Pep mixture, at a NP concentration of 5 mg/mL. After this mixture was agitated on a nutating mixer at 4° C. for 40 min, the NPs were centrifuged at 15,000 rcf for 2 min at 4° C., and then isolated from the solution using the DynaMag. The supernatant was collected and saved as the flow-through (F) fraction. The isolated NPs were then washed three times with a wash buffer (50 mM Tris, pH 7.5, 300 mM NaCl; 0.20 mL/mg NP) following the same centrifugation and magnetic isolation steps to remove unbound, non-specific proteins. To elute the bound cTnI, 500 μL of 200 mM glycine hydrochloride buffer (pH 2.2) was added. After centrifugation and magnetic isolation, the resulting supernatant was collected as the elution fraction (E). All protein fractions (L, F, and E) were desalted prior to MS analysis using a 10 kDa MWCO filter (Amicon, 0.5 mL, cellulose, MilliporeSigma) and buffered exchanged using 0.2% formic acid in nanopure water. To evaluate the enrichment performance of the functionalized NPs, all collected fractions (L, F, and E) were equally loaded (500 ng), separated using a polyacrylamide gel (12.5%), and stained with SYPRO Ruby (Thermo Fisher Scientific, Inc., Rockford, Ill., USA) fluorescent dye. The gel was imaged using a ChemiDoc™ MP Imaging System (170-8280; Bio-Rad, Hercules, Calif., USA).
cTnI enrichment using the agarose solid-support platform from human cardiac sarcomere extracts. mAb or peptide was conjugated to the agarose beads following the manufacturer's recommendations and blocked with a 1.0 M ethanolamine solution, pH 7.4. Protein loading mixture (L) was incubated with 500 μL of mAb or peptide-conjugated agarose beads in a disposable affinity column for 40 min on a nutating mixer at 4° C. After incubation, the supernatant was collected and saved as the flow-through (F) fraction. The agarose beads were then washed three times with 1 mL of 50 mM Tris pH 7.4 and 300 mM NaCl to elute unbound proteins. Subsequently, the bound proteins were eluted using four equal fractions of 500 μL of 200 mM glycine hydrochloride, pH 2.2. The four elution fractions (E) were pooled and all protein fractions (L, F, and E) were desalted prior to MS analysis using a molecular weight cutoff filter (Amicon, 0.5 mL, cellulose, MilliporeSigma) and buffered exchanged using 0.2% formic acid in nanopure water.
cTnI enrichment using the NP-Pep from human serum. Serum loading mixtures were prepared by serially spiking in human sarcomeric protein extracts into 200 μL of serum (10 mg protein) from human male AB plasma and diluting the serum mixture to a final volume of 1 mL using a buffered solution of 50 mM Tris pH 7.4 and 1.0 M LiCl. The concentrations of cTnI spiked into serum samples were measured using a cTnI AccuBind ELISA kit (Monobind Inc., Lake Forest, Calif., USA). The cTnI spiked human serum loading mixture (L) was incubated with 5 mg of peptide-functionalized NPs in a 2 mL Eppendorf Lo-Bind tube. After this mixture was agitated on a nutating mixer at 4° C. for 40 min, the NPs were centrifuged at 15,000 rcf for 2 min at 4° C., and then isolated from the solution using a DynaMag. The supernatant was collected and saved as the flow-through (F) fraction. The NPs were then washed three times with 1 mL of 50 mM Tris pH 7.4 and 300 mM NaCl following the same centrifugation and magnetic isolation steps to remove unbound, non-specific proteins. To elute the bound cTnI, 500 μL of 200 mM glycine hydrochloride buffer (pH 2.2) was added. After centrifugation and magnetic isolation, the resulting supernatant was collected as the elution fraction (E). All protein fractions (L, F, and E) were desalted prior to MS analysis using a 10 kDa MWCO filter (Amicon, 0.5 mL, cellulose, MilliporeSigma) and buffer exchanged using 0.2% formic acid in nanopure water.
cTnI enrichment using the agarose solid-support platform from human serum. The serum loading mixture was prepared in the same way as in the NP serum enrichment protocol and the mAb or peptide conjugate agarose beads were prepared as previously described. The serum loading mixture (L) was added to 500 μL of mAb or peptide-conjugated agarose beads in a disposable affinity column, and agitated for 40 min on a nutating mixer at 4° C. After incubation, the supernatant was collected and saved as the flow-through (F) fraction. The agarose beads were then washed three times with 1 mL of 50 mM Tris pH 7.4 and 300 mM NaCl to elute unbound proteins. Subsequently, the bound proteins were eluted using four equal fractions of 500 μL of 200 mM glycine hydrochloride, pH 2.2. The four elution fractions (E) were pooled and all protein fractions (L, F, and E) were desalted prior to MS analysis using a 10 kDa MWCO filter (Amicon, 0.5 mL, cellulose, MilliporeSigma) and buffered exchanged using 0.2% formic acid in nanopure water.
Comparison of the cTnI enrichment performance of the NP-Pep platform against the agarose-mAb or agarose-Pep platform from human cardiac tissue extracts. The enrichment workflow was performed in a cold room held at 4° C. to minimize possible artifactual protein modifications, such as oxidation. NP-Control (NP-BAPTES), NP-Pep (Fe3O4-BAPTES-Peptide), Agarose-Control beads (no coupling), Agarose-Pep beads, and Agarose-mAb beads were incubated with sarcomeric extract obtained from completely de-identified healthy donor heart tissue, diseased heart tissue of dilated cardiomyopathy (DCM), and post-mortem heart tissue containing 306 ng cTnI, 482 ng cTnI, and 465 ng cTnI in the protein extract, respectively. cTnI values were determined by ELISA quantification as previously described. The loading mixture (L), flow through (F), and elution mixture (E) were collected, desalted using a 10 kDa MWCO filter, and buffer exchanged prior to SDS-PAGE gel analysis or LC-MS analysis as described in the tissue enrichment protocol. The methods described here correspond to the data presented in
Comparison of the cTnI enrichment performance of the NP-Pep platform against the agarose-mAb or agarose-Pep platform from human serum spiked with cTnI. The enrichment workflow was performed in a cold room held at 4° C. to minimize possible artifactual protein modifications, such as oxidation. NP-Control (NP-BAPTES),NP-Pep (Fe3O4-BAPTES-Peptide), Agarose-Control beads (no coupling), Agarose-Pep beads, and Agarose-mAb beads were incubated with in a protein loading mixture containing human male AB serum (10 mg) and sarcomeric tissue extract obtained from healthy donor heart tissue, diseased heart tissue of DCM, and post-mortem heart tissue containing 306 ng cTnI, 482 ng cTnI, and 465 ng cTnI in the protein extract, respectively. cTnI values were determined by ELISA quantification as previously described. The loading mixture (L), flow through (F), and elution mixture (E) were collected, desalted using a 10 kDa MWCO, and buffer exchanged prior to SDS-PAGE gel analysis or LC-MS analysis as described in the tissue enrichment protocol. The methods described here correspond to the data presented in
Typical reverse phase chromatography (RPC) procedure. Reverse phase chromatography (RPC) was performed with a nanoACQUITY UPLC system (Waters; Milford, Mass., USA). Mobile phase A (MPA) contained 0.2% formic acid in nanopure water, and mobile phase B (MPB) contained 0.2% formic acid in 50:50 acetonitrile: isopropanol. Prior to injection, protein samples were desalted by washing through 10 kDa MWCO filters using 0.2% formic acid in nanopure water six times. For each injection, 500 ng of desalted protein sample was loaded onto a BIOshell A400 C4, 3.4 μm, 15 cm×200 μm capillary column. The column was placed in a column heater set at 60° C. with a constant 3 μL/min flow rate. The RPC gradient consisted of the following concentrations of MPB: 10% MPB at 0 min, 10% at 5 min, 65% at 45 min, 90% at 50 min, held at 90% until 55 min, adjusted back to 10% at 55.1 min, and held at 10% until 60 min. Each run was 60 min long.
Top-down MS analysis. Samples eluted from RPC separation were ionized using a CaptiveSpray source (Bruker Daltonics, Bremen, Germany) into a MaXis II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) for online LC-MS and LC-MS/MS experiments. End plate offset and capillary voltage were set at 500 and 4000 V, respectively. The nebulizer was set to 0.3 bar, and the dry gas flow rate was 4.0 L/min at 200° C. The quadruple low mass cutoff was set to 500 m/z during MS and 200 m/z during MS/MS. Mass range was set to 200-3000 m/z and spectra were acquired at 1 Hz for LC-MS runs. All data were collected with OtofControl 3.4 (Bruker Daltonics), analyzed and processed in DataAnalysis 4.3 (Bruker Daltonics). Maximum Entropy algorithm (Bruker Daltonics) was used to deconvolute all mass spectra with the resolution set to 80,000. Sophisticated Numerical Annotation Procedure (SNAP) peak-picking algorithm (quality factor: 0.4; signal-to-noise ratio (S/N): 3.0; intensity threshold: 500) was applied to determine the monoisotopic mass of all detected ions. Fragmentation ion lists consisting of monoisotopic mass, intensity and charge were generated from DataAnalysis 4.3, and were subsequently converted to MSAlign files. TopPIC was used to search against the Uniprot-Swissprot human database which was released on Nov. 9, 2018 and contains 20395 protein sequences. Fragment mass tolerance was set to 15 ppm. All identifications were validated with statistically significant P and E values (<0.01) and satisfactory numbers of assigned fragment (>10). Tandem mass spectra were output from the DataAnalysis software and analyzed using MASH Suite Pro software (Cai et al., 2016, Molecular & Cellular Proteomics, 15(2): 703-714). The spectra were deconvoluted with a signal-to-noise ratio of 3 and a cutoff fit score of 60%. All the program-processed data were manually validated to obtain accurate sequence and PTM information. Chromatograms in
Statistical analysis. All statistical data were presented as the mean±standard error of the mean (SEM). Student's t-test was performed between group comparisons to evaluate the statistical significance of variance for the validation of the simultaneous quantification of protein expression and modification changes. Differences among means were considered significant at p<0.05. All error bars shown in the figures were based on SEMs.
Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.
When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Additionally, the end points in a given range are to be included within the range. In the disclosure and the claims, “and/or” means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.
One of ordinary skill in the art will appreciate that starting materials, device elements, analytical methods, mixtures and combinations of components other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Headings are used herein for convenience only.
All publications referred to herein are incorporated herein to the extent not inconsistent herewith. Some references provided herein are incorporated by reference to provide details of additional uses of the invention. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.
This invention was made with government support under GM117058 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/066011 | 12/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62949869 | Dec 2019 | US |
