Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

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. Molecular & Cellular

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…xVis 58 , xiNET 59 , ProXL 60 , and CLMSVault 61 are examples of recently developed software that permit visualization of cross-linking data as networks of connected residues. In addition, CLMSVault 61 facilitates mapping of cross-links in the context of three-dimensional structures, similar to ProXL 60 and XlinkDB 2.0 62 , which also function as public repositories for cross-linking data.…”

“…xVis 58 , xiNET 59 , ProXL 60 , and CLMSVault 61 are examples of recently developed software that permit visualization of cross-linking data as networks of connected residues. In addition, CLMSVault 61 facilitates mapping of cross-links in the context of three-dimensional structures, similar to ProXL 60 and XlinkDB 2.0 62 , which also function as public repositories for cross-linking data. While most mapping tools utilize Euclidean distances to determine cross-link length, Xwalk and Jwalk have been developed to determine solvent-accessible surface distances (SASD) for cross-links, the length of the shortest path between two amino acids with respect to protein volume 63,64 .…”

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

“…It has been previously shown that immunoaffinity and mass spectrometric methodologies can be combined for the effective screening of hybridoma supernatants against the native forms of antigens [14] or for elucidation of antigens bound to mAbs of unknown specificity [19]. In the present study, we utilized a mass spectrometer (Q-Exactive Plus) to design PRM experiments (Additional file 2: Figure S1) and rapidly screen the monoclonal antibodies [14, 19, 20]. A potential limitation of the current protocol is perhaps the inaccuracy of antigen quantification in some cases.…”

Searching for prognostic biomarkers for small renal masses in the urinary proteome

“…All samples were analyzed in triplicate and run on the Q‐Exactive Plus (Thermo Fisher Scientific) mass spectrometer. Limit of detection and limit of quantification were calculated, as described previously . Heavy‐labeled peptide amount was optimized relative to the light peptide amount observed in the unfractionated urine pool so that the ratio of light and heavy peptide was close to 1.…”