FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2472989101> ?p ?o ?g. }

• W2472989101 endingPage "2876" @default.
• W2472989101 startingPage "2863" @default.
• W2472989101 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. 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. The human tissue kallikrein-related peptidases (KLKs) 1The abbreviations used are:KLKsTissue kallikrein-related peptidasesAUCArea under the curveCVFCervicovaginal fluidELISAEnzyme-linked immunosorbent assayFPKMFragments per kilobase of transcript per million mapped readsFWHMFull width at half maximumPRMParallel reaction monitoringROCReceiver operating characteristicSPSeminal plasmaSRMSelected reaction monitoringSWSweat. are a large family of 15 closely related serine proteases with trypsin- or chymotrypsin-like activities. Kallikreins exhibit important similarities both at the gene and protein level (1.Sotiropoulou G. Pampalakis G. Diamandis E.P. Functional roles of human kallikrein-related peptidases.J. Biol. Chem. 2009; 284: 32989-32994Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The genes are localized to chromosome 19q13.4, forming the largest contiguous cluster of proteases within the human genome (2.Yousef G.M. Kopolovic A.D. Elliott M.B. Diamandis E.P. Genomic overview of serine proteases.Biochem. Biophys. Res. Commun. 2003; 305: 28-36Crossref PubMed Scopus (49) Google Scholar). The enzymes are initially secreted as inactive zymogens and subsequently activated by removal of a short N-terminal pro-sequence (3.Yousef G.M. Diamandis E.P. The new human tissue kallikrein gene family: structure, function, and association to disease.Endocr. Rev. 2001; 22: 184-204PubMed Scopus (0) Google Scholar). KLKs are expressed in many tissues, including steroid hormone-producing or hormone-dependent tissues and are responsible for the coordination of various physiological functions including regulation of blood pressure (4.Pathak M. Wong S.S. Dreveny I. Emsley J. Structure of plasma and tissue kallikreins.Thromb Haemost. 2013; 110: 423-433Crossref PubMed Scopus (48) Google Scholar), neuronal plasticity (5.Attwood B.K. Bourgognon J.M. Patel S. Mucha M. Schiavon E. Skrzypiec A.E. Young K.W. Shiosaka S. Korostynski M. Piechota M. Przewlocki R. Pawlak R. Neuropsin cleaves EphB2 in the amygdala to control anxiety.Nature. 2011; 473: 372-375Crossref PubMed Scopus (148) Google Scholar), semen liquefaction (6.Michael I.P. Pampalakis G. Mikolajczyk S.D. Malm J. Sotiropoulou G. Diamandis E.P. Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression.J. Biol. Chem. 2006; 281: 12743-12750Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), skin desquamation (7.Borgono C.A. Michael I.P. Komatsu N. Jayakumar A. Kapadia R. Clayman G.L. Sotiropoulou G. Diamandis E.P. A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation.J. Biol. Chem. 2007; 282: 3640-3652Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) and inflammation (8.Hollenberg M.D. Oikonomopoulou K. Hansen K.K. Saifeddine M. Ramachandran R. Diamandis E.P. Kallikreins and proteinase-mediated signaling: proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer.Biol. Chem. 2008; 389: 643-651Crossref PubMed Scopus (54) Google Scholar), and thus represent attractive diagnostic and therapeutic targets (9.Prassas I. Eissa A. Poda G. Diamandis E.P. Unleashing the therapeutic potential of human kallikrein-related serine proteases.Nat. Rev. Drug Discov. 2015; 14: 183-202Crossref PubMed Scopus (162) Google Scholar). Aberrant levels of some KLKs were observed in tissues, blood serum and proximal fluids of cancer patients, particularly in cases of adenocarcinomas derived from steroid hormone-regulated tissues, and were correlated with the course of disease. For instance, concurrent up-regulation of multiple KLKs has been observed in ovarian carcinoma (10.Yousef G.M. Polymeris M.E. Yacoub G.M. Scorilas A. Soosaipillai A. Popalis C. Fracchioli S. Katsaros D. Diamandis E.P. Parallel overexpression of seven kallikrein genes in ovarian cancer.Cancer Res. 2003; 63: 2223-2227PubMed Google Scholar). It has also been shown for breast, prostate, and testicular cancers that KLKs may facilitate neoplastic progression through promotion of cell proliferation and metastasis (11.Dorn J. Bayani J. Yousef G.M. Yang F. Magdolen V. Kiechle M. Diamandis E.P. Schmitt M. Clinical utility of kallikrein-related peptidases (KLK) in urogenital malignancies.Thromb. Haemost. 2013; 110: 408-422Crossref PubMed Scopus (24) Google Scholar). Based on this evidence, kallikrein-related peptidases have been extensively studied for their potential as biomarkers of various malignancies (12.Borgono C.A. Diamandis E.P. The emerging roles of human tissue kallikreins in cancer.Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (553) Google Scholar, 13.Yousef G.M. Polymeris M.E. Grass L. Soosaipillai A. Chan P.C. Scorilas A. Borgono C. Harbeck N. Schmalfeldt B. Dorn J. Schmitt M. Diamandis E.P. Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer.Cancer Res. 2003; 63: 3958-3965PubMed Google Scholar, 14.Diamandis E.P. Okui A. Mitsui S. Luo L.Y. Soosaipillai A. Grass L. Nakamura T. Howarth D.J. Yamaguchi N. Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma.Cancer Res. 2002; 62: 295-300PubMed Google Scholar, 15.Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Human kallikrein 14: a new potential biomarker for ovarian and breast cancer.Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar, 16.Diamandis E.P. Yousef G.M. Human tissue kallikreins: a family of new cancer biomarkers.Clin. Chem. 2002; 48: 1198-1205Crossref PubMed Scopus (210) Google Scholar). Tissue kallikrein-related peptidases Area under the curve Cervicovaginal fluid Enzyme-linked immunosorbent assay Fragments per kilobase of transcript per million mapped reads Full width at half maximum Parallel reaction monitoring Receiver operating characteristic Seminal plasma Selected reaction monitoring Sweat. High-quality ELISAs are now available for the majority of KLKs, except KLKs 1, 9, 12, and 15. Because of their specificity, sensitivity, accuracy, high throughput, and relative simplicity, ELISAs have been used for decades to measure KLKs in biological and clinical samples (17.Shaw J.L. Diamandis E.P. Distribution of 15 human kallikreins in tissues and biological fluids.Clin. Chem. 2007; 53: 1423-1432Crossref PubMed Scopus (311) Google Scholar). Rapid evolution of biological mass spectrometry provided powerful alternatives to immunoassays for unambiguous identification and accurate quantification of proteins in clinical samples (18.Blankley R.T. Fisher C. Westwood M. North R. Baker P.N. Walker M.J. Williamson A. Whetton A.D. Lin W. McCowan L. Roberts C.T. Cooper G.J. Unwin R.D. Myers J.E. A label-free selected reaction monitoring workflow identifies a subset of pregnancy specific glycoproteins as potential predictive markers of early-onset pre-eclampsia.Mol. Cell. Proteomics. 2013; 12: 3148-3159Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 19.Shi T. Gao Y. Quek S.I. Fillmore T.L. Nicora C.D. Su D. Zhao R. Kagan J. Srivastava S. Rodland K.D. Liu T. Smith R.D. Chan D.W. Camp 2nd, D.G. Liu A.Y. Qian W.J. A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum.J. Proteome Res. 2014; 13: 875-882Crossref PubMed Scopus (50) Google Scholar). Selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) assays have high specificity, dynamic range of six orders of magnitude, and limits of detection (LOD) and quantification (LOQ) in the low ng/ml level. Unlike multiplex ELISA, which allows for the detection of up to 25 analytes in a single assay (20.Tighe P.J. Ryder R.R. Todd I. Fairclough L.C. ELISA in the multiplex era: potentials and pitfalls.Proteomics Clin. Appl. 2015; 9: 406-422Crossref PubMed Scopus (244) Google Scholar), multiplex SRM assays facilitate measurements of tens to hundreds of proteins in a single run without compromising assay sensitivity and accuracy (21.Percy A.J. Chambers A.G. Yang J. Hardie D.B. Borchers C.H. Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.Biochim. Biophys. Acta. 2014; 1844: 917-926Crossref PubMed Scopus (120) Google Scholar, 22.Drabovich A.P. Pavlou M.P. Dimitromanolakis A. Diamandis E.P. Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay.Mol. Cell. Proteomics. 2012; 11: 422-434Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23.Burgess M.W. Keshishian H. Mani D.R. Gillette M.A. Carr S.A. Simplified and efficient quantification of low-abundance proteins at very high multiplex via targeted mass spectrometry.Mol. Cell. Proteomics. 2014; 13: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24.Huttenhain R. Soste M. Selevsek N. Rost H. Sethi A. Carapito C. Farrah T. Deutsch E.W. Kusebauch U. Moritz R.L. Nimeus-Malmstrom E. Rinner O. Aebersold R. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.Sci. Transl. Med. 2012; 4: 142ra194Crossref Scopus (208) Google Scholar). Here, we present multiplatform targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs with limits of quantification in the low ng/ml range in clinical samples. The analytical performance of the SRM assays was evaluated by measuring absolute levels of endogenous KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum samples and by comparison to the performance of selected ELISAs. Finally, using the whole set of SRM, immuno-SRM and sandwich immunoassays, we investigated KLK4 and unequivocally resolved earlier disputes about its isoform identity in seminal plasma and its levels in prostate cancer. Iodoacetamide, dithiothreitol, acetonitrile, formic acid, and sequencing grade modified trypsin were purchased from Sigma-Aldrich (Oakville, ON, Canada). RapiGest SF surfactant was purchased from Waters (Milford, MA). Quantified & Stable Isotope Labeled Peptides (SpikeTides™_TQL) were obtained from JPT Peptide Technologies GmbH (Berlin, Germany). The recombinant KLK4 was purified through a two-step purification protocol and was used as an immunogen for the production of monoclonal antibodies in mice, as previously described (25.Obiezu C.V. Shan S.J. Soosaipillai A. Luo L.Y. Grass L. Sotiropoulou G. Petraki C.D. Papanastasiou P.A. Levesque M.A. Diamandis E.P. Human kallikrein 4: quantitative study in tissues and evidence for its secretion into biological fluids.Clin. Chem. 2005; 51: 1432-1442Crossref PubMed Scopus (56) Google Scholar). Clone 10F4.1G6 was used as a capture antibody for the analysis of seminal plasma and blood serum samples with immunoenrichment-SRM and ELISA. Rabbit polyclonal anti-KLK4 antibodies were used as detection antibodies. All biological samples were collected from individuals of different ethnic background with an informed consent. Sample collection was approved by the institutional review boards of Mount Sinai Hospital (approval #08–117-E and, #16–0137-E) and University Health Network (# 09–0830-AE). Semen samples were collected by masturbation into sterile collection cups. Following liquefaction for 1 h at room temperature, semen samples were centrifuged at 16,000 rpm for 30 min at 4 °C three times to separate seminal plasma (SP) from cells and cellular components, and were stored at −80 °C until further use. Ten SP samples were obtained from healthy men (median age 35 years old) prior to vasectomy, 21 SPs from men with biopsy-confirmed prostate cancer (median age 62) and 20 SPs from men with negative biopsy outcome (median age 62). In addition, blood serum samples were obtained from 36 men with biopsy-confirmed prostate cancer (serum PSA > 4 ng/ml, median age 63), 22 men with negative biopsy (serum PSA > 4 ng/ml, median age 61), and 3 healthy men (serum PSA < 1 ng/ml, median age 36). Five cervico-vaginal fluid (CVF) samples were collected from nonpregnant women (median age 30) in flexible cups that are worn internally, around the cervix. Ten sweat (SW) samples were collected from five men and five women (median age 28) after physical exercise in wood fiber wipes (KimWipes, Kimberly-Clark, Mississauga, ON, Canada). A 20 ml syringe was used to squeeze the fluid out of the wipes. All samples were centrifuged at 16,000 × g for 30 min to ensure complete removal of cells and cellular debris. Before trypsin digestion, the total protein content of each sample was measured using the Pierce BCA protein assay (Thermo Scientific, Mississauga, ON, Canada). Twenty micrograms of total protein per sample were subjected to proteomic sample preparation. To prepare for digestion, denaturation of proteins and reduction of disulfide bonds were achieved by treatment of samples with 0.1% RapiGest SF and 10 mm dithiothreitol at 65 °C for 15 min. After reduction, the samples were alkylated with 20 mm iodoacetamide in the dark at room temperature for 40 min. Heavy isotope-labeled peptides with a quantitation tag (500 fmoles) were added to all samples prior to trypsin digestion. Digestion (1:20, trypsin/total protein) was performed overnight at 37 °C. RapiGest SF was cleaved with 1% trifluoroacetic acid and removed with centrifugation at 16,000 × g for 15 min. C18 Bond Elute OMIX tips (10 μl; Agilent Technologies, Mississauga, ON, Canada) were used for desalting and concentration of tryptic peptides (final solution in 5% acetonitrile and 0.1% formic acid). Shotgun and PRM experiments were performed on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). The tryptic peptides were loaded on a 3 cm long C18 (5 μm) trap precolumn (i.d. 200 μm) via EASY-nLC 1000 pump (Thermo Scientific) at 8 μl/min. Mobile phases included 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). Peptides were separated with a 15 cm long C18 (3 μm) analytical column (i.d. 75 μm) with a 8 μm tip (New Objective, Woburn, MA) with a 22 min gradient elution at 350 nL/min flow rate. A five-step gradient was used: 1% to 14% of buffer B for 1 min, 14% to 40% for 11 min, 40% to 65% for 2 min, 65% to 100% for 1 min, and 100% for 7 min. Shotgun experiments included one full MS1 scan followed by 12 data-dependent MS2 scans. In-source collision induced dissociation (CID) was set to 0.0 eV, and the ion transfer capillary temperature was 275 °C. MS transitions in the orbitrap were acquired with 70,000 resolving power at 200 m/z. Automatic Gain Control (AGC) for MS1 acquisition was set to 3 × 106 with a maximum injection time of 100 ms. AGC target for the data dependent MS2 scans acquisition was set to 105 with a maximum injection time of 50 ms and the normalized collision energy of 27. The scan range was 250 to 1500 m/z, microscans were set to 1 for both MS1 and MS2, charge state screening was enabled and unassigned, and +1 charge states were excluded from MS2 activation. The performance settings for the PRM assays were the following: in-source CID was set to 3.0 eV, MS transitions in the orbitrap were acquired with 17,500 resolving power at 200 m/z, AGC target was set to 3 × 106 with a maximum injection time of 100 ms, the isolation window was set to 1.0 m/z, the normalized collision energy was set to 23 and scan times were set to 100 ms. The development of SRM assays and the analysis of clinical samples were performed using a TSQ QuantivaTM triple quadrupole mass spectrometer (Thermo Scientific), as previously described (26.Drabovich A.P. Pavlou M.P. Schiza C. Diamandis E.P. Dynamics of protein expression reveals primary targets and secondary messengers of estrogen receptor alpha signaling in MCF-7 breast cancer cells.Mol. Cell. Proteomics. 2016; 15: 2093-2107Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Polarity was set to positive, ion transfer tube temperature was 300°C, and the CID argon pressure was 1.5 mTorr. The resolution settings of the first and third quadrupoles were 0.2 and 0.7 Da FWHM, respectively (comparable to the “high” and “unit” resolution of the QTRAP 6500 quadrupole-ion trap mass spectrometer). High resolution in the first quadrupole facilitated exclusion of potentially interfering ions, thus improving selectivity in the complex biological matrices. Scan times were set to 10 ms. Optimized SRM method was also tested in a QTRAP 6500 quadrupole-ion trap mass spectrometer (AB Sciex, Concord, ON, Canada). Declustering and entrance potentials were set to 150 and 10 V, respectively. Resolutions for both the first quadrupole and the ion trap were set to “unit,” and scan times were 15 ms. To facilitate identification of proteotypic peptides, we aimed at identifying five to seven peptides that would result in intense and reproducible MS1 signal. For the selection of top peptides, we used tryptic digests of 15 recombinant KLKs previously expressed in our lab in prokaryotic (27.Kishi T. Grass L. Soosaipillai A. Shimizu-Okabe C. Diamandis E.P. Human kallikrein 8: immunoassay development and identification in tissue extracts and biological fluids.Clin. Chem. 2003; 49: 87-96Crossref PubMed Scopus (58) Google Scholar), mammalian (HEK293) (28.Kishi T. Soosaipillai A. Grass L. Little S.P. Johnstone E.M. Diamandis E.P. Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids.Clin. Chem. 2004; 50: 709-716Crossref PubMed Scopus (42) Google Scholar, 29.Shaw J.L. Grass L. Sotiropoulou G. Diamandis E.P. Development of an immunofluorometric assay for human kallikrein 15 (KLK15) and identification of KLK15 in tissues and biological fluids.Clin. Biochem. 2007; 40: 104-110Crossref PubMed Scopus (19) Google Scholar, 30.Diamandis E.P. Yousef G.M. Soosaipillai A.R. Grass L. Porter A. Little S. Sotiropoulou G. Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.Clin. Biochem. 2000; 33: 369-375Crossref PubMed Scopus (122) Google Scholar) or yeast (Pichia pastoris) (13.Yousef G.M. Polymeris M.E. Grass L. Soosaipillai A. Chan P.C. Scorilas A. Borgono C. Harbeck N. Schmalfeldt B. Dorn J. Schmitt M. Diamandis E.P. Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer.Cancer Res. 2003; 63: 3958-3965PubMed Google Scholar, 15.Borgono C.A. Grass L. Soosaipillai A. Yousef G.M. Petraki C.D. Howarth D.H. Fracchioli S. Katsaros D. Diamandis E.P. Human kallikrein 14: a new potential biomarker for ovarian and breast cancer.Cancer Res. 2003; 63: 9032-9041PubMed Google Scholar, 31.Kapadia C. Chang A. Sotiropoulou G. Yousef G.M. Grass L. Soosaipillai A. Xing X. Howarth D.H. Diamandis E.P. Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay.Clin. Chem. 2003; 49: 77-86Crossref PubMed Scopus (59) Google Scholar) expression systems. The LC-MS/MS raw data were analyzed using the Proteome Discoverer™ software (Thermo Scientific, version 1.4.1.14) with SEQUEST search algorithm and the human UniProtKB/Swiss-Prot database (HUMAN_sProt-07092014; 20,209 entries). The mass tolerances for precursor and fragment ions were set to 7 ppm and 0.02 Da, respectively. The Proteome Discoverer decoy database and Percolator algorithm were used to compute the number of false positive protein identifications based on the Posterior Error Probabilities. The false discovery rate based on Q-values was set to 1%. The following parameters were set for the search: (1) digestion enzyme: trypsin; (2) maximum allowance for miss-cleavages: 1; (3) oxidation of methionine and tryptophan, as well as N-terminal peptide acetylation were set as variable modifications, whereas cysteine carbamidomethylation was set as a static modification. Unmodified peptides as well as peptides with proline were preferred, if possible. Peptides with abundant modifications based on three analytical replicates that included the digestion step (MS1 area of forms with methionine oxidation, glutamine and asparagine deamidation, N-terminal protein acetylation, or missed cleavage sites >10% of total MS1 area) were selected for further investigation. All peptides were searched with the protein Basic Local Alignment Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure that the selected peptides were unique for each KLK. Raw mass spectrometry data and Proteome Discoverer output files with peptide and protein identifications were deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/login) with the data set identifier PXD003324 and the following credentials: Username: [email protected]; Password: b4Ge5SYs. To facilitate accurate protein quantification, we aimed at experimental testing of three peptides for each KLK. First, in silico digestion of all proteins was performed using the Skyline Targeted Proteomics Environment v3.1.0.7382 (MacCoss Lab Software, Seattle, WA). The top three peptides for each protein were selected based on our full MS data dependent MS/MS identification data and were subsequently confirmed with the SRM atlas (www.srmatlas.org). Because of the presence of N terminus cysteine in one of the selected peptides for KLK5, the pyro-carbamidomethyl modified form of this peptide was also selected for quantification. In the second step of method development, the selected peptides were included into each of 15 unscheduled survey PRM assays designed for Q Exactive Plus (supplemental Table S1). Comparison of LC retention time for shotgun and PRM gradients was used as another indication of identity of selected peptides (32.Drabovich A.P. Jarvi K. Diamandis E.P. Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay.Mol. Cell. Proteomics. 2011; 10 (M110 004127)Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The ten most intense and selective transitions per peptide were selected. In the third step, 10 transitions per selected peptide were included into each of 15 unscheduled targeted mass spectrometry methods on QTRAP 6500 quadrupole-ion trap and TSQ QuantivaTM triple quadrupole mass spectrometers. Based on the relative intensities, the most intense peptides and the three most intense and reproducible transitions per peptide were selected for SRM assays (supplemental Tables S2 and S3). Calibration curves for all 45 peptides (derived by digestion of 15 recombinant KLKs) were built, and a single peptide per each KLK was selected based on its limit of detection, quantification, linearity and coefficient of variation (supplemental Table S4). The limit of detection was defined as the lowest analyte concentration distinguished from the background (S/N≥3). The limit of quantification was defined as the lowest concentration measured with CV≤20% within the linear range of the calibration curve. In order to exclude possible interferences, ensure correct identity of each peak and facilitate absolute quantification of each protein, heavy isotope-labeled peptide internal standards with a quantifying tag were synthesized for all 15 KLKs. Following peptide synthesis, the quantifying tag (serine-alanine-[3-nitro]tyrosine-glycine) ensured accurate peptide quantification using UV absorption at 350 nm and was readily cleaved by trypsin, thus accounting for digestion efficiency. The area of the endogenous peptide was compared with that of the heavy peptide, and their ratio was used to calculate the absolute concentration of the endogenous light peptide. In the fourth step of method development, 32 heavy and light peptides and 96 transitions were included into multiplex unscheduled PRM or SRM assays. Selectivity of transitions and possible interferences in each sample matrix were assessed. Finally, 15 KLKs, 32 peptides, and 96 transitions where scheduled within 2 min intervals in a single multiplex scheduled SRM assay (supplemental Figs. S1–S3). Scan times were optimized to ensure acquisition of at least 20 points per peak. Precursor-to-product transitions were provided in the supplemental Tables S5–7. SRM and PRM raw mass spectrometry data were deposited to the Peptide Atlas repository (http://www.peptideatlas.org/PASS/PASS00777) with the dataset identifier PASS00777 and the following credentials: Username: PASS00777; Password: VU2858zr. Peptides were separated on a C18 analytical column with a 22 min gradient elution and subsequently detected by TSQ QuantivaTM mass spectrometer with a nanoelectrospray ionization source. The total method duration, including sample pickup and loading, pre-column and column equilibration was 48 min, thus resulting in a throughput of 30 runs per day. The performance of the nanoLC-MS platforms was assessed at the beginning of each day and every six runs thereafter using injections of 10 μl of 1 fmol/μl bovine serum albumin (BSA) solution. Thus, the throughput was 12 clinical samples per day in duplicates (24 injections). Repeatability of the method and the system stability were estimated by the coefficient of variation (CV%). Carryover ranged between 0.2–3.1% and was estimated by blank injections. Linearity of the assay was studied by spiking increasing amounts of the heavy labeled peptides" @default.
• W2472989101 created "2016-07-22" @default.
• W2472989101 creator A5044981509 @default.
• W2472989101 creator A5049664994 @default.
• W2472989101 creator A5056642098 @default.
• W2472989101 creator A5066989467 @default.
• W2472989101 creator A5084226800 @default.
• W2472989101 date "2016-09-01" @default.
• W2472989101 modified "2023-10-18" @default.
• W2472989101 title "Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays" @default.
• W2472989101 cites W1579539545 @default.
• W2472989101 cites W1964937857 @default.
• W2472989101 cites W1968313287 @default.
• W2472989101 cites W1973321737 @default.
• W2472989101 cites W1974001790 @default.
• W2472989101 cites W1977911881 @default.
• W2472989101 cites W1982149110 @default.
• W2472989101 cites W1985043450 @default.
• W2472989101 cites W1986448517 @default.
• W2472989101 cites W1993977352 @default.
• W2472989101 cites W1997135204 @default.
• W2472989101 cites W1997360117 @default.
• W2472989101 cites W1998993932 @default.
• W2472989101 cites W1999981067 @default.
• W2472989101 cites W2004101774 @default.
• W2472989101 cites W2009054305 @default.
• W2472989101 cites W2015248164 @default.
• W2472989101 cites W2018093134 @default.
• W2472989101 cites W2020670793 @default.
• W2472989101 cites W2021443328 @default.
• W2472989101 cites W2024572769 @default.
• W2472989101 cites W2026334766 @default.
• W2472989101 cites W2032643022 @default.
• W2472989101 cites W2041820065 @default.
• W2472989101 cites W2045168608 @default.
• W2472989101 cites W2047322941 @default.
• W2472989101 cites W2057622930 @default.
• W2472989101 cites W2061877464 @default.
• W2472989101 cites W2076841583 @default.
• W2472989101 cites W2084905965 @default.
• W2472989101 cites W2095270540 @default.
• W2472989101 cites W2099086346 @default.
• W2472989101 cites W2099387726 @default.
• W2472989101 cites W2100339207 @default.
• W2472989101 cites W2102948567 @default.
• W2472989101 cites W2103227382 @default.
• W2472989101 cites W2104728010 @default.
• W2472989101 cites W2108401997 @default.
• W2472989101 cites W2110958540 @default.
• W2472989101 cites W2116529176 @default.
• W2472989101 cites W2116556918 @default.
• W2472989101 cites W2124995176 @default.
• W2472989101 cites W2125366716 @default.
• W2472989101 cites W2131730392 @default.
• W2472989101 cites W2132058414 @default.
• W2472989101 cites W2136663003 @default.
• W2472989101 cites W2136828548 @default.
• W2472989101 cites W2140145660 @default.
• W2472989101 cites W2146501048 @default.
• W2472989101 cites W2149620854 @default.
• W2472989101 cites W2153569860 @default.
• W2472989101 cites W2154511147 @default.
• W2472989101 cites W2159286617 @default.
• W2472989101 cites W2161863487 @default.
• W2472989101 cites W2162129864 @default.
• W2472989101 cites W2167387123 @default.
• W2472989101 cites W2172811803 @default.
• W2472989101 cites W2314170505 @default.
• W2472989101 cites W2324380049 @default.
• W2472989101 doi "https://doi.org/10.1074/mcp.m115.057695" @default.
• W2472989101 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5013304" @default.
• W2472989101 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/27371727" @default.
• W2472989101 hasPublicationYear "2016" @default.
• W2472989101 type Work @default.
• W2472989101 sameAs 2472989101 @default.
• W2472989101 citedByCount "43" @default.
• W2472989101 countsByYear W24729891012017 @default.
• W2472989101 countsByYear W24729891012018 @default.
• W2472989101 countsByYear W24729891012019 @default.
• W2472989101 countsByYear W24729891012020 @default.
• W2472989101 countsByYear W24729891012021 @default.
• W2472989101 countsByYear W24729891012022 @default.
• W2472989101 countsByYear W24729891012023 @default.
• W2472989101 crossrefType "journal-article" @default.
• W2472989101 hasAuthorship W2472989101A5044981509 @default.
• W2472989101 hasAuthorship W2472989101A5049664994 @default.
• W2472989101 hasAuthorship W2472989101A5056642098 @default.
• W2472989101 hasAuthorship W2472989101A5066989467 @default.
• W2472989101 hasAuthorship W2472989101A5084226800 @default.
• W2472989101 hasBestOaLocation W24729891011 @default.
• W2472989101 hasConcept C162356407 @default.
• W2472989101 hasConcept C181199279 @default.
• W2472989101 hasConcept C185592680 @default.
• W2472989101 hasConcept C3020728648 @default.
• W2472989101 hasConcept C43617362 @default.
• W2472989101 hasConcept C52853521 @default.
• W2472989101 hasConcept C55493867 @default.
• W2472989101 hasConcept C70721500 @default.

• next