Plasma Proteome Institute

Washington, DC, United States

Plasma Proteome Institute

Washington, DC, United States
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Razavi M.,University of Victoria | Pope M.E.,University of Victoria | Soste M.V.,University of Victoria | Soste M.V.,ETH Zurich | And 4 more authors.
Journal of Immunological Methods | Year: 2011

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10. min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma. © 2010.

Boja E.S.,U.S. National Institutes of Health | Jortani S.A.,University of Louisville | Ritchie J.,Emory University | Hoofnagle A.N.,University of Washington | And 8 more authors.
Clinical Chemistry | Year: 2011

BACKGROUND: Clinical proteomics presents great promise in biology and medicine because of its potential for improving our understanding of diseases at the molecular level and for detecting disease-related biomarkers for diagnosis, prognosis, and prediction of therapeutic responses. To realize its full potential to improve clinical outcome for patients, proteomic studies have to be well designed, from biosample cohorts to data and statistical analyses. One key component in the biomarker development pipeline is the understanding of the regulatory science that evaluates diagnostic assay performance through rigorous analytical and clinical review criteria. CONTENT: The National Cancer Institute's Clinical Proteomic Technologies for Cancer (CPTC) initiative has proposed an intermediate preclinical "verification" step to close the gap between protein-based biomarker discovery and clinical qualification. In collaboration with the US Food and Drug Administration (FDA), the CPTC network investigators recently published 2 mock submission review documents, first-of-their-kind educational materials that may help the scientific community interested in developing products for the clinic in understanding the likely analytical evaluation requirements for multiplex protein technology- based diagnostic tests. CONCLUSIONS: Building on this momentum, the CPTC continues with this report its collaboration with the FDA, as well as its interactions with the AACC and the Centers for Medicare and Medicaid Services, to further the understanding of regulatory requirements for approving multiplex proteomic platform - based tests and analytically validating multiple analytes. © 2011 American Association for Clinical Chemistry.

Whiteaker J.R.,Fred Hutchinson Cancer Research Center | Zhao L.,Fred Hutchinson Cancer Research Center | Abbatiello S.E.,The Broad Institute of MIT and Harvard | Burgess M.,The Broad Institute of MIT and Harvard | And 8 more authors.
Molecular and Cellular Proteomics | Year: 2011

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Parker C.E.,University of Victoria | Pearson T.W.,University of Victoria | Anderson N.L.,Plasma Proteome Institute | Borchers C.H.,University of Victoria
Analyst | Year: 2010

This review reports on the current and emerging technologies for the use of mass-spectrometry-based proteomics in clinical applications. © 2010 The Royal Society of Chemistry.

Anderson N.L.,Plasma Proteome Institute | Anderson N.L.,SISCAPA Assay Technologies | Razavi M.,University of Victoria | Pearson T.W.,SISCAPA Assay Technologies | And 4 more authors.
Journal of Proteome Research | Year: 2012

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R 2 values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers. © 2012 American Chemical Society.

Kuhn E.,The Broad Institute of MIT and Harvard | Whiteaker J.R.,Fred Hutchinson Cancer Research Center | Mani D.R.,The Broad Institute of MIT and Harvard | Jackson A.M.,University of Victoria | And 9 more authors.
Molecular and Cellular Proteomics | Year: 2012

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies.We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 μl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and < 14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Drake P.M.,University of California at San Francisco | Cho W.,Purdue University | Li B.,Buck Institute for Age Research | Prakobphol A.,University of California at San Francisco | And 5 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT: In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies - multiple reaction monitoring and lectin-antibody arrays - as potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY: The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses. © 2009 American Association for Clinical Chemistry.

Whiteaker J.R.,Fred Hutchinson Cancer Research Center | Zhao L.,Fred Hutchinson Cancer Research Center | Anderson L.,Plasma Proteome Institute | Paulovich A.G.,Fred Hutchinson Cancer Research Center
Molecular and Cellular Proteomics | Year: 2010

There is an urgent need for quantitative assays in verifying and validating the large numbers of protein biomarker candidates produced in modern "-omics" experiments. Stable isotope standards with capture by anti-peptide antibodies (SISCAPA) has shown tremendous potential to meet this need by combining peptide immunoaffinity enrichment with quantitative mass spectrometry. In this study, we describe three significant advances to the SISCAPA technique. First, we develop a method for an automated magnetic bead-based platform capable of high throughput processing. Second, we implement the automated method in a multiplexed SISCAPA assay (nine targets in one assay) and assess the performance characteristics of the multiplexed assay. Using the automated, multiplexed platform, we demonstrate detection limits in the physiologically relevant ng/ml range (from 10 μl of plasma) with sufficient precision (median coefficient of variation, 12.6%) for quantifying biomarkers. Third, we demonstrate that enrichment of peptides from larger volumes of plasma (1 ml) can extend the limits of detection to the low pg/ml range of protein concentration. The method is generally applicable to any protein or biological specimen of interest and holds great promise for analyzing large numbers of biomarker candidates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Klee E.W.,Rochester College | Bondar O.P.,Rochester College | Goodmanson M.K.,Rochester College | Trushin S.A.,Rochester College | And 3 more authors.
American Journal of Clinical Pathology | Year: 2014

Objectives: Harmonization of prostate-specific antigen (PSA) immunoassays is important for good patient care. The specificity of the antibodies used to detect circulating PSA could cause differences in the PSA measurements. Methods: We used mass spectrometry (MS) to quantitate the concentration of five peptides cleaved from trypsin digestion of PSA and compared these measurements with six automated immunoassays. Linear regression and a mixed-effects model were used to analyze the results. Results: PSA measurements from the immunoassays and the five MS peptide assays were highly correlated (R2 > 0.99), but the recovery of the World Health Organization standard and the regression slopes differed across assays. The same relative patterns of immunoassay differences were seen in comparing their results with each of the five MS peptide measurements from different parts of the circulating PSA molecules. Conclusions: Mass spectrometry quantitation of peptides derived from trypsin digestion of immune-extracted PSA could be used to harmonize PSA immunoassays. © American Society for Clinical Pathology.

Klee E.W.,Rochester College | Bondar O.P.,Rochester College | Goodmanson M.K.,Rochester College | Trushin S.A.,Rochester College | And 4 more authors.
Archives of Pathology and Laboratory Medicine | Year: 2014

Context. - Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays. Objective. - To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays. Design. - Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer. Results. - The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis. Conclusions. - This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays. © 2014, College of American Pathologists. All rights reserved.

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