Abstract
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.
Highlights
From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada; ʈLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
Development of Targeted Mass Spectrometry Assays—Our method development was based on selection of proteotypic peptides using recombinant proteins and experimental LCMS/MS, LC-parallel reaction monitoring (PRM), and LC-Selected reaction monitoring (SRM) data
Multiplex PRM and SRM assays for all 15 KLKs and for three different mass spectrometry platforms (Q Exactive Plus, TSQ Quantiva, and QTRAP 6500) were developed
Summary
From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, Ontario, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada; ʈLunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. 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. Quantification of Kallikreins by Mass Spectrometry for breast, prostate, and testicular cancers that KLKs may facilitate neoplastic progression through promotion of cell proliferation and metastasis [11]. Based on this evidence, kallikrein-related peptidases have been extensively studied for their potential as biomarkers of various malignancies [12,13,14,15,16]. Unlike multiplex ELISA, which allows for the detection of up to 25 analytes in a single assay [20], multiplex SRM assays facilitate measurements of tens to hundreds of proteins in a single run without compromising assay sensitivity and accuracy [21,22,23,24]
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