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Skates S.J.,Massachusetts General Hospital | Gillette M.A.,The Broad Institute of MIT and Harvard | LaBaer J.,Arizona State University | Carr S.A.,The Broad Institute of MIT and Harvard | And 17 more authors.
Journal of Proteome Research | Year: 2013

Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor, and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC) with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step toward building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research. © 2013 American Chemical Society. Source

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. Source

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. Source

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. Source

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. Source

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