Automated Microchromatography Enables Multiplexing of

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Automated Micro-chromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-based Assays Paul J Ippoliti, Eric Kuhn, D. R. Mani, Lola Fagbami, Hasmik Keshishian, Michael W Burgess, Jacob D. Jaffe, and Steven A. Carr Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00946 • Publication Date (Web): 18 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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Analytical Chemistry

Automated Microchromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-based Assays

Paul J. Ippoliti; Eric Kuhn*; D. R. Mani; Lola Fagbami^; Hasmik Keshishian; Michael W. Burgess; Jacob D. Jaffe; Steven A. Carr*

Broad Institute of MIT and Harvard, Cambridge, MA 02142 ^

Current address: Harvard School of Public Health, Boston, MA 02115

*

To whom correspondence should be addressed:

Steven A. Carr, Email: [email protected] Eric Kuhn, Email: [email protected]

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Abbreviations: AAA: amino acid analysis MRM or SRM: multiple reaction monitoring or selected reaction monitoring SID-MRM-MS: Stable Isotope Dilution Multiple Reaction Monitoring Mass Spectrometry fMRM: (chromatographic) fractionation MRM-MS IP: immunoprecipitation SISCAPA: Stable Isotope Standards Capture with Anti-Peptide Antibodies Immuno-MRM-MS (iMRM): Immunoprecipitation Multiple Reaction Monitoring Mass Spectrometry

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Abstract: Immunoaffinity enrichment of peptides coupled with analysis by stable isotope dilution multiple reaction mass spectrometry has been shown to have analytical performance and detection limits suitable for many biomarker verification studies and biological applications. Prior studies have shown that anti-peptide antibodies can be multiplexed up to 50 in a single assay without significant loss of performance. Achieving higher multiplex levels is relevant to all studies involving precious biological material as this minimizes the amount of sample that must be consumed to measure a given set of analytes and reduces the assay cost per analyte. Here we developed automated methods employing the Agilent AssayMAP Bravo microchromatography platform and used these methods to characterize the performance of immunoaffinity enrichment of peptides up to multiplex levels of 172. Median capture efficiency for the target peptides remained high (88%) even at levels of 150-plex and declined to 70% at 172-plex compared to antibody performance observed at standard lower multiplex levels (n = 25). Subsequently, we developed and analytically characterized a multiplexed immuno-MRM-MS assay (n = 110) and applied it to measure candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction. The median lower limit of detection of all peptides was 71.5 amol/µL (nM) and the coefficient of variation (CV) was less than 15% at the lower limit of quantification. The results demonstrate that high multiplexed immuno-MRM-MS assays are readily achievable using the optimized sample processing and peptide capture methods described here.

Keywords: Targeted mass spectrometry; anti-peptide rabbit polyclonal antibody; protein biomarkers; plasma proteins; multiple reaction monitoring mass spectrometry; selected reaction monitoring; multiplexed; microchromatography

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Introduction: Stable isotope-dilution (SID) coupled with targeted mass spectrometry is now a widely accepted approach for verification of candidate protein biomarkers and, increasingly, for precise measurement of peptides, modified peptides and proteins in biological studies (see 1–3 for recent reviews and references therein). Multiple reaction monitoring mass spectrometry (MRM-MS, also known as selected reaction monitoring (SRM)) on triple quadrupole mass spectrometers has been the most widely applied technology for these applications1–3. SID-MRM-MS assays have demonstrated performance suitable to detect and quantify proteins in plasma 4–14 with detection limits at or below 1 ng/mL in plasma when samples are first pre-processed by multi-dimensional chromatography 15–17. MRM-based assays have also been developed to measure peptides/proteins in cell lysates 18–20 and tissues 21–23. Unlike immunoassays, MRM-based assays can be highly multiplexed, with recent examples achieving up to 400-plex in a single analysis 15. Specifications have been derived to classify the fit-for-purpose performance of these assays 24 and publicly accessible portals are being created to share assays and their performance metrics25. In order to detect analytes present at low-to-sub nanogram/mL levels 26,27, numerous methods for pre-enrichment of analytes prior to SID-MRM-MS have been explored1 including the removal of highly abundant plasma proteins and separation of digested peptides into less complex fractions by HPLC 12,13,16–18,21,28,29. These techniques have been used individually or in combination to improve LOD of mass spectrometry-based assays. While immunoaffinity depletion of abundant proteins and peptide fractionation are of proven value for increasing depth of detection in complex matrices, these methods also increase the complexity of sample handling, introduce potential sources of pre-analytical variability, and significantly increase the amount of time required to execute a reproducible assay across a large number of samples for a large number of analytes. To help alleviate the bottleneck of sample processing while maintaining low detection limits and measurement precision for targeted peptide quantification, Anderson et al. 30 introduced the use of immunoaffinity capture of peptides as a pre-enrichment step prior to MRM-MS. In this method, antibodies (Abs) are raised 4


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against tryptic peptides and then used to immunoprecipitate these same peptides from trypsin digests of samples together with their spiked-in stable isotope labeled versions for quantification by MRM-MS. This method, called Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA, also referred to as immuno-MRM (iMRM)), can provide greater than 1000-fold analyte enrichment without depletion of abundant proteins or subsequent fractionation of protein digests 31–37 with equivalent demonstrated performance when multiplexed 38. Unlike conventional immunoassays, iMRM assays are not subject to interferences from heterophilic antibodies 39,40. The SISCAPA technology has been successfully applied for verification of candidate biomarkers and in pharmacodynamics studies 31,34–36 and has been shown to have interlaboratory assay performance similar to that of direct and fractionation MRM assays 37. Recently, Whiteaker et al., developed iMRM assays on a Kingfisher magnetic bead-handling robot to monitor the DNA damage response using a mixture of polyclonal antibodies raised against 44 distinct phosphorylated peptides 36. These Abs successfully immunoprecipitated the phosphorylated forms of each of the peptides and, in some cases also captured the unmodified peptide, resulting in a total of 67 peptides assayed 36. While this is the highest multiplexed iMRM assay developed to date, the demonstrated multiplexing levels of iMRM still falls far short of multiplex levels demonstrated for MRM assays 15,19. Here we describe development of a new automated method to increase iMRM multiplex levels to greater than 150. The approach takes advantage of the capabilities of the Agilent AssayMAP Bravo microchromatography platform equipped with 96 individual syringes, each fitted with a Protein G AssayMAP cartridge for antibody enrichment of peptides41. Multiplex iMRM assay levels up to 172 were developed to test the effect of multiplexing on assay performance. A multiplexed iMRM assay (n = 110) was then configured, analytically characterized and subsequently applied to assay candidate protein biomarkers of cardiovascular disease in plasma of patients undergoing planned myocardial infarction (PMI). Using this assay, we discovered proteins with measurable concentration trends that had not previously been associated with PMI.

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Experimental Section: Peptide Standards, Rabbit Anti-peptide Polyclonal Antibodies, and Plasma – 226 peptides specific to 126 proteins were identified and selected using previously described methods and criteria 28,33 (Table S1). “Heavy” peptides and “light” peptides were synthesized, formulated, quantified by amino acid analysis (AAA) and evaluated and stored as described in the Supporting Information. Anti-peptide polyclonal antibodies were generated from rabbits (Epitomics, New England Peptide) and evaluated as previously described 42 (see Supporting Information). Plasma for assay development was acquired from unidentified human subjects (Bioreclamation Inc.) and treated as non-clinical samples. Plasma samples were obtained from patients at Mass General Hospital undergoing planned myocardial infarction (PMI) with alcohol ablation for the treatment of hypertrophic obstructive cardiomyopathy (HOCM) 12–14. All protocols for blood collection were approved by the Massachusetts General Hospital Institutional Review Board, and all subjects gave written informed consent. Preparation of Peptide Curves for iMRM – A bulk preparation of digested plasma was resuspended in 1X PBS/0.03% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) (see Plasma Digestion section in the Supporting Information) and light peptides were added to a concentration of 100 fmol/10 µL digested plasma. This solution was used as the diluent for response curve preparation using heavy peptides as the surrogate for unlabeled analytes 43,44. Five hundred femtomoles of heavy peptide per 10 µL digested plasma were added to the highest concentration point and serially diluted over 7 concentrations (Reverse Curve). A blank (0 fmol heavy peptide, 100 fmol light peptide) was also prepared. Each concentration point was prepared in singlicate in the final antibody enrichment volume (200 µL) and aliquoted in triplicate into KingFisher™ 250 microtiter plates and frozen at -80 oC until use. Automated Peptide Immunoaffinity Enrichment on AssayMAP Bravo – Plates containing digested and desalted plasma from either reverse curves (n = 24 samples) or PMI patient time course samples (n = 20 samples) were thawed. Reverse curve samples at 200 µL per well were placed directly onto AssayMAP Bravo. PMI patient samples containing the equivalent of 30 µL plasma 6


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per time point per patient were divided into 3 replicates of 10 µL and aliquoted into separate plates for single replicate antibody enrichment on 3 separate days. All wash reagents were freshly prepared, dispensed into a 384 well plate (Figure S8) and placed on the Bravo AssayMAP platform with the other reagent plates as shown in Figure S9. Antibody crosslinked Protein G cartridges were prepared as described in Supporting Information and primed with 100 µL 1X PBS at 300 µL/min and equilibrated with 50 µL 1X PBS at 25 µL/min. After priming, 200 uL of each sample were loaded onto cartridges at 2 µL/min. Cartridges were washed 3 times, once each with 50 µL of the following sequence of solutions at 10 µL/min: 5X PBS, 1X PBS and 50% Acetonitrile/0.2 M ammonium bicarbonate. After washing, bound peptides were eluted with 35 µL 0.01 N Hydrochloric acid at 5 µL/min. After elution, cartridges were re-equilibrated with 50 µL 1 M Tris-HCl pH 8.0 at 10 µL/min and 50 µL 1X PBS at 25 µL/min. Prior to storage at 4 °C, cartridges were conditioned with 50 µL 1X PBS/0.01% sodium azide. NanoLC-MRM-MS Analysis Reverse curves and enriched PMI patient plasma were analyzed on a TSQ Quantiva triple quadrupole mass spectrometer installed with a Nanospray Flex source (Thermo Fisher Scientific) and Easy-nLC 1000 system. Ion source was set to positive ion mode with capillary temperature of 300oC, spray voltage of 2000 and sweep gas set to 0. Easy-nLC 1000 system was primed with mobile phase A (3% acetonitrile/0.1% formic acid), mobile phase B (90% acetonitrile/0.1% formic acid). Samples were injected (2 µL, 20% of enriched sample) onto a 0.075 mm ID PicoFrit (New Objective) column pulled to a 10 um emitter and custom-packed to 10 cm with 3 µm 200Å C18-AQ Reprosil beads (Dr. Maisch). The LC gradient was 5% B for 3 min, 5% B to 40% B in 50 min, 40% B to 90 % B in 2.3 min. Three transitions were monitored per peptide by scheduled MRM (Table S1) using a 6 minute RT window and a 1.5 s cycle time. Collision energies were optimized over 4 steps, 2.5 V per step in smaller unscheduled batches of less than 500 transitions per batch. Extracted Ion chromatograms (XIC) of all transition ions were integrated using a Skyline document (Skyline daily version 2.6. https://brendanx7



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uw1.gs.washington.edu/labkey/project/home/software/Skyline/begin.view) 45. Integrated peaks were manually inspected to confirm proper integration and detection of the transitions for the corresponding light and heavy peptides. Integrated peaks were further processed using QuaSAR46 in Skyline (also available at: http://genepattern.broadinstitute.org/) and the peptide molar concentration (fmol/µL or uM), regression analysis results (slope, intercept and R2)47 and limits of detection (LOD) and quantification (LOQ) using a modified Linnet method48 were determined. The concentration of each detected protein (ng/mL) was calculated from the peptide molar concentration and the protein molecular weight (in Table S2 and UniProt, http://www.uniprot.org/) using the following equation: (1) = [(fmol/µL)] x [Molecular Weight fg/fmol] ÷ 1000]

Results and Discussion: The primary goal of this study was to explore and optimize methods for increasing the number of antibodies that can be multiplexed into a single iMRM assay without significantly compromising assay performance. The overall process employed is illustrated in Figure 1. The binding capacity of the AssayMAP Protein G cartridges used to bind the anti-peptide antibodies was determined to be greater than 275 µg IgG per cartridge using a commercial source of IgG isolated from nonimmunized rabbits (data not shown). At this binding capacity we estimated that iMRM assay multiplex levels of 140 to 300 could be achieved using our standard amount of 1 µg to 2 µg of polyclonal antibodies per capture. After preliminary screening, a set of 172 anti-peptide antibodies from our inventory were selected for multiplex optimization studies (see Supporting Information) based on the following properties: capture efficiencies for the target peptides of greater than 5%; co-elution of all monitored transitions; peak heights of greater than 10,000 counts-per-second for at least one transition. The anti-peptide antibodies used here derived from a number of on-going studies in the laboratory employing iMRM assays to verify plasma-based candidate biomarkers of human breast cancer, cardiovascular disease and the acute phase 8


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response. Five multiplex panels each comprised of free, unbound antibodies with similar capture efficiencies were then constructed from these 172 working antibodies (Figure 1). Specifically, the 25 antibodies with the highest capture efficiencies (highest H/L ratios) were combined into the first multiplex “bundle”. Additional multiplex “bundles” were then constructed by mixing the first set of 25 antibodies with the next highest ranking set of antibodies (n = 25) to create a multiplex bundle of 50 and so on until multiplex bundles of 25, 50, 100, 150, and 172 were constructed. The five antibody bundles of increasing multiplex level were loaded onto Protein G AssayMAP cartridges and chemically crosslinked using the Bravo robot as described in Supporting Information. In parallel, the antibodies were loaded and crosslinked to cartridges in discrete multiplexes of 20-25 Abs to assess performance. The performance of increasing multiplex levels of iMRM was then evaluated using the Agilent AssayMAP Bravo microchromatography platform (Figure 1A). Aliquots of digested plasma (see Supporting Information) from a healthy donor pool containing 200 fmol heavy peptide standards were aspirated and dispensed twice over a 4 h capture period onto the antibody cartridges (see Methods). Following capture, the antibodies were washed and bound heavy synthetic and unlabeled endogenous peptides (if present) were eluted. Light synthetic peptides were added to the eluate in equimolar amounts to the pre-capture heavy peptide spike levels and analyzed by LC-MRM-MS. Heavy-to-light peak area ratios were calculated for each peptide to determine the capture efficiency. In order to determine the effect of antibody multiplexing, the antibody capture efficiency in each multiplex bundle was divided by the capture efficiency determined in the parallel capture experiment (n = 25 antibodies) as described in the Supporting Information. The median of these normalized capture efficiencies was plotted for each multiplex level (Figure S11) to assess antibody performance. Capture efficiency was consistently close to 100% for multiplex bundles approaching 100 (median capture efficiency = 97%). Median capture efficiency declined to 88% at 150 which we still considered adequate. At the highest level (n = 172 Abs), corresponding to an antibody cartridge load of 350 µg, peptide capture efficiency was reduced to 70% of that observed at multiplex level of 25, most likely due to having exceeded the

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capacity of the Protein G cartridge which we estimated earlier to be 300 µg of rabbit IgG. Based on these results, we selected 120 antibodies with the highest peptide recoveries for further study. Evaluation of a Multiplex of 120 immuno-MRM-MS (iMRM) Assays using Peptide Reverse Curves The 120 antibodies with the best capture efficiencies were crosslinked onto protein G AssayMAP cartridges on the Bravo system and used for immunoaffinity enrichment as described in Supporting Information. Response curves were prepared by spiking a fixed concentration of light peptide standards into digested plasma and then spiking heavy peptide standards spanning a concentration range of 3 orders of magnitude (see Methods). After immunoaffinity enrichment was performed in process triplicate, the captured heavy and light peptides in each sample were analyzed by LC-MRM-MS in singlicate (see Methods and Supporting Information). From these response curves, CV, LOD, LOQ and linearity were determined for all antibody/peptide pairs using the software tool QuaSAR as described in Methods. The performance of each antibody was classified into three categories based on the response curves. Examples of each category are shown in Figure S12. Antibodies placed into category A had response curves that were linear throughout the concentration range, with good agreement observed for all transitions (e.g NME.YGASWTAEK in Figure S12). Over 95 of the 120 antibodies were category A antibodies. The linear range of category B antibodies was generally smaller with noticeable differences in response between the 3 transitions especially in the lower concentration range, which resulted in a higher LOD (e.g. DBI.WDAWNELK in Figure S12). Category A and B antibodies have similar slopes, but Category A antibodies have detection limits below 0.4 fmol/µL (µM) (Table S2). There were 10 category C antibodies. These antibodies were defined by having poor linearity and slopes, with average R2 less than 0.25 and average slopes < 0.6, or > 1.4 (e.g. FMOD.IPPVNTNLENLYLQGNR in Figure S12). Category C antibodies were removed and the response curves of the remaining 110 peptides were subsequently analyzed.

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Response curves for each category A and B antibody were analyzed and the LOD, LOQ and CVs for the peptides were used to evaluate the overall performance of the assays. Median CVs and LODs are summarized in Figure 2; individual performance of each antibody/peptide pair is presented in Table S2. Category A and B peptides/antibodies generated assays with good overall reproducibility, with imprecision of less than 20 % at all concentrations down to ca. 100 amol/µL (nM). Interquartile range for all detectable transitions was between 2 – 50 fmol/µL (µM) and increased proportionally at the lower end of the concentration curve to 70 amol/µL (nM). The large interquartile range observed for the blank sample (0 fmol/µL heavy peptides added, Figure 2A) was due to the presence of only baseline background ions contributing to the signal. The observed LOD for the 110 peptides analyzed ranged from 14 amol/µL (nM) to 3200 amol/µL (nM), with a median LOD of 71.5 amol/µL (nM) in plasma (Figure 2B and Table S2). The median CV for these peptides was 8.9% near the LOD (1.9 fmol/µL (µM)) and 3.5% at the LOQ (16.7 fmol/µL (µM)) (Table S2). These findings compare favorably to the values of CV and LOD/LOQ that have been reported for smaller multiplex iMRM assays where magnetic beads were used for immunoaffinity enrichment of peptides from digested plasma 9,14,37,42,49. The demonstrated assay performance for this high multiplex iMRM assay meets recommended performance criteria for Tier 2 assays 24. Application of the High Multiplex iMRM Assay to Quantify Peptides in PMI Patient Samples We next assessed the performance and utility of the high multiplex iMRM assay (n = 110 Abs) for measuring relative changes in endogenous protein levels in plasma collected from patients undergoing the planned myocardial infarction procedure (PMI) (Figure 1B). Plasma samples were collected from five cardiovascular patients before PMI and at 3 time points post PMI. The time points selected for this study were chosen based on previous discovery studies in our laboratory that were designed to discover predictive biomarkers of myocardial infarction by measuring plasma protein concentration changes in the same patient over a specified timecourse during the PMI procedure 13,14. Plasma samples were digested in singlicate for each time point and patient using a Bravo LT liquid handling robot (as described in the Supporting Information), 11



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spiked with heavy peptide standards and then divided into three equal parts for immunoaffinity enrichment in process triplicate. Of the 110 working antibodies, 46 peptides derived from 41 protein targets were detected and quantified in PMI plasma samples. To understand the reference ranges of protein concentration accessible using a high multiplex iMRM assay, we generated a protein concentration box and whisker plot analogous in design to those previously reported for plasma proteins by Anderson 26 and Hortin 27 based on results published using different protein assays (ELISA, LC-MS, etc.) obtained on different plasma samples. The interquartile ranges and median protein concentrations for the 46 peptides (from 41 proteins) measured in the same sample using the same assay format (iMRM) are plotted in Figure 3 (Table S3). The range of protein concentrations measured spanned over 4 orders of magnitude. At the highest end of the range we quantified SERPINC1.VAEG and PROS1.IETI with concentrations of 10-30 µg/mL. At the lower end of the concentration range, 3 proteins PRDX4.QITL, PI3.GPVS and TNNI3.NITE were measured at or below 10 ng/mL. In those cases where more than one peptide was measured for a given protein (e.g., VWF, MBL2, SAA1, SPON1, KRT19) the peptides from the same protein showed similar ranges in concentration. Peptides with large interquartile ranges represent those proteins whose concentration varies over the PMI time course or between patients. For example the biological CV of SAA1 (peptides SFFS and GPGG), was over 100%, whereas the measured analytical CV from the response curve data was less than 10 %. Individual time course trend plots (data not shown) identified that the wide CV range observed was not due to the PMI procedure, but rather one patient that had a much higher, yet reproducible, SAA1 concentration throughout the time course.

Use of High Multiplex iMRM assays for Discovery As previously noted, the anti-peptide antibodies used in the present study came from a range of ongoing prior studies in our laboratory. Only 8 of the 46 peptides we quantified using the 110plex assay had been selected for iMRM-MS assay development and were monitored in our 12


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previous PMI cardiovascular verification studies 13,14. To determine whether any of the 38 newly measured and quantified peptides were also regulated during the PMI time course we performed a moderated F-test (http://genepattern.broadinstitute.org/ ) on all 46 peptides detected. Using a pvalue threshold of 0.05, we identified 14 peptides (13 proteins) with significant fold-changes during the PMI time course (Table S4). To visualize the differences in observed protein concentration trends, we clustered the F-test results using GENE-E (http://www.broadinstitute.org/cancer/software/GENE-E/ ), and generated a heat map (Figure 4). Three distinct trend clusters were identified when the standardized log ratio relative to baseline for each peptide were plotted (Figure 4). The 5 peptides in trend Cluster 1 were up relative to baseline at 10 min and 1 h, then decreased significantly by 4 h, while the 5 peptides in Cluster 2 were lower relative to baseline at 10 min, increased to their highest level at 1 h, then returned to near baseline levels after 4 h. The concentrations of the 4 peptides in trend Cluster 3 increased continuously and reached their highest levels after 4 h. Six of the eight peptides measured in our earlier iMRM PMI study (AEBP1.DTPV, MYL3.ALGQ, SPON1.AQWP and SPON1.VEGD in Cluster 1 and peptides FHL1.AIVA and FHL1.AIVA in Cluster 2 (Table S4) were found to change significantly in the present study and to exhibit similar temporal concentration trends to those we observed previously using a different set of patient samples 14. The concentrations of the other two peptides, TAGLN2.ENFQ and FSTL1.IQVD, did not change during PMI time course in the patient samples used in the present study. Variability in the behavior of these two peptides/proteins is also consistent with our prior observations 14. In addition, six peptides/proteins not previously targeted for analysis were observed to change over the PMI time course (CLIC1.GFTI – cluster 1, DKK3.DQDG, VCAN.LGEP, VWF.AVSP – cluster 2 and IFGBP2.GECW, PPIC.TVEN – cluster 3). These results suggest that highly multiplexed MRM assays may also be a discovery mode to probe plasma or other types of sample matrices in addition to being used for candidate verification.

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Conclusions: We have demonstrated that iMRM multiplex levels of greater than 150 can be achieved using sample processing on the AssayMAP robotic system with Protein G cartridges. Additional improvements in performance are anticipated. Titrating the amount of Ab used to immunoaffinity enrich each target 50 (e.g. 0.1 – 4 µg) could allow the level of multiplexing to be increased without loss of performance. The amount of antibody used per assay could be reduced further by generating monoclonal antibodies which have advantages over polyclonal antibodies 51

, including higher binding affinities and ease of reproduction. With these advancements, we

estimate that multiplex levels could be increased to 200 to 300 on the current AssayMAP protein G cartridges. In addition to reducing the cost of analysis per analyte, increased multiplexing and use of automation as demonstrated using this panel of antibodies makes it possible to simultaneously screen or verify larger numbers of biological candidate proteins without compromising assay precision.

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References: (1) (2) (3) (4) (5)

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Acknowledgments: This work was supported in part through NCI Clinical Proteomics Tumor Analysis Consortium initiative grant U24CA160034 to S.A.C. and by a Thought Leader Award and gift to S.A.C. from the Agilent Technologies Foundation and Agilent Technologies, Inc. We thank Zach Van Den Heuvel and Steve Murphy, Agilent Technologies, Inc. for their help and consultation with the Bravo LT, Bravo AssayMAP and VWorks. We also thank Leslie Gaffney, Broad Institute, for her help with the TOC Graphic.

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Supporting Information: Supplementary Experimental: Details of peptide QC, antibody generation, automated plasma digestion and immunoaffinity enrichment. Supplementary Figures: Diagrams and platemap configurations of Bravo LT and Bravo AssayMAP and results of higher multiplexing experiment and assignment of antibody categories based on response curve performance. Supplementary References: References for methods for bulk digest of plasma with trypsin, antibody generation and MRM-MS data analysis. Supplementary Tables: List of proteins, gene names, peptides, antibodies and MRM transition ions, assay performance, F-test and results of QuaSAR analysis used to plot PMI plasma reference range.

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Figure Legends: Figure 1. Overview of the development and application of highly multiplexed iMRM assays. A. Polyclonal antibodies were screened and evaluated in multiplex bundles ranging from 25 to 172 using protein G cartridges and the AssayMAP Bravo (see Methods and Supporting Information). A subset of the antibodies that performed well was selected and evaluated as a multiplex of 110. B. Performance of the multiplexed iMRM assay (n = 110 Abs) was assessed by analysis of plasma from patients undergoing treatment for cardiovascular disease. Hierarchical clustering identified proteins with statistically significant changes in protein concentration. Figure 2. Assessment of a Multiplex of 110 iMRM assays using Reverse Curves. Reverse curves were generated in digested plasma by serial dilution of 120 heavy peptides from 50 fmol/µL (µM) to 0 fmol/µL (µM) and immunoaffinity enriched with antibodies crosslinked onto AssayMAP protein G cartridges. Category C antibodies were removed prior to regression analysis using QuaSAR46. A) Assessment of assay reproducibility using the median CV of the 3 transitions monitored for 110 category A and B peptides (see Table S2). B) The median lower limit of detection for the capture and analysis of 110 peptides in undiluted plasma was 71.5 amol/µL (nM). Black dots represent outliers. Figure 3. Reference range of proteins detected in PMI patient plasma. Concentration of proteins detected in PMI patient plasma listed for each peptide (e.g. TNNI3.NITEIADLTQK) for all four time points ranked from highest median concentration to lowest on a log10 scale. Figure 4. Cluster analysis to determine types of statistically significant trends observed in the PMI time course. F-test analysis of the 46 peptides (41 proteins) detected in PMI plasma using the multiplexed iMRM assay (n = 110 Abs) identified 14 peptides (13 proteins) with significant trends (p value < 0.05) (Table S4). Hierarchal clustering identified three types of concentration trends in the first 4 hours of a PMI.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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for TOC only

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Automated Microchromatography Enables Multiplexing of

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