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Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

ABSTRACT: BACKGROUND:For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT:The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

SUBMITTER: Hoofnagle AN

PROVIDER: S-EPMC4830481 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

Hoofnagle Andrew N AN Whiteaker Jeffrey R JR Carr Steven A SA Kuhn Eric E Liu Tao T Massoni Sam A SA Thomas Stefani N SN Townsend R Reid RR Zimmerman Lisa J LJ Boja Emily E Chen Jing J Crimmins Daniel L DL Davies Sherri R SR Gao Yuqian Y Hiltke Tara R TR Ketchum Karen A KA Kinsinger Christopher R CR Mesri Mehdi M Meyer Matthew R MR Qian Wei-Jun WJ Schoenherr Regine M RM Scott Mitchell G MG Shi Tujin T Whiteley Gordon R GR Wrobel John A JA Wu Chaochao C Ackermann Brad L BL Aebersold Ruedi R Barnidge David R DR Bunk David M DM Clarke Nigel N Fishman Jordan B JB Grant Russ P RP Kusebauch Ulrike U Kushnir Mark M MM Lowenthal Mark S MS Moritz Robert L RL Neubert Hendrik H Patterson Scott D SD Rockwood Alan L AL Rogers John J Singh Ravinder J RJ Van Eyk Jennifer E JE Wong Steven H SH Zhang Shucha S Chan Daniel W DW Chen Xian X Ellis Matthew J MJ Liebler Daniel C DC Rodland Karin D KD Rodriguez Henry H Smith Richard D RD Zhang Zhen Z Zhang Hui H Paulovich Amanda G AG

Clinical chemistry 20160101 1

<h4>Background</h4>For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently ...[more]

PMID: 26719571

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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

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

Publications

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

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