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Prediction of high-responding peptides for targeted protein assays by mass spectrometry.

ABSTRACT: Protein biomarker discovery produces lengthy lists of candidates that must subsequently be verified in blood or other accessible biofluids. Use of targeted mass spectrometry (MS) to verify disease- or therapy-related changes in protein levels requires the selection of peptides that are quantifiable surrogates for proteins of interest. Peptides that produce the highest ion-current response (high-responding peptides) are likely to provide the best detection sensitivity. Identification of the most effective signature peptides, particularly in the absence of experimental data, remains a major resource constraint in developing targeted MS-based assays. Here we describe a computational method that uses protein physicochemical properties to select high-responding peptides and demonstrate its utility in identifying signature peptides in plasma, a complex proteome with a wide range of protein concentrations. Our method, which employs a Random Forest classifier, facilitates the development of targeted MS-based assays for biomarker verification or any application where protein levels need to be measured.

SUBMITTER: Fusaro VA

PROVIDER: S-EPMC2753399 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Prediction of high-responding peptides for targeted protein assays by mass spectrometry.

Fusaro Vincent A VA Mani D R DR Mesirov Jill P JP Carr Steven A SA

Nature biotechnology 20090125 2

Protein biomarker discovery produces lengthy lists of candidates that must subsequently be verified in blood or other accessible biofluids. Use of targeted mass spectrometry (MS) to verify disease- or therapy-related changes in protein levels requires the selection of peptides that are quantifiable surrogates for proteins of interest. Peptides that produce the highest ion-current response (high-responding peptides) are likely to provide the best detection sensitivity. Identification of the most ...[more]

PMID: 19169245

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Project description:Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

Dataset Information

Prediction of high-responding peptides for targeted protein assays by mass spectrometry.

Publications

Prediction of high-responding peptides for targeted protein assays by mass spectrometry.

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