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• W2900768108 abstract "TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm–oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101 and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm–oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower (p = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction. TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm–oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101 and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm–oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower (p = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction. Recent -omics studies identified 1,079 human genes with exclusive expression in testis (1Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. Sivertsson Å. Kampf C. Sjöstedt E. Asplund A. Olsson I. Edlund K. Lundberg E. Navani S. Szigyarto C.A. Odeberg J. Djureinovic D. Takanen J.O. Hober S. Alm T. Edqvist P.H. Berling H. Tegel H. Mulder J. Rockberg J. Nilsson P. Schwenk J.M. Hamsten M. von Feilitzen K. Forsberg M. Persson L. Johansson F. Zwahlen M. von Heijne G. Nielsen J. Pontén F. Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (7240) Google Scholar). While function of many of testis-specific proteins is not known, it may be assumed that these proteins have unique and specialized roles in spermatogenesis and fertilization. Mutations, natural knockouts, or deleterious single nucleotide variations in testis-specific genes could lead to spermatogenesis arrest, reduced sperm concentration or motility, abnormal sperm morphology, or impaired sperm–oocyte interaction (2Nuti F. Krausz C. Gene polymorphisms/mutations relevant to abnormal spermatogenesis.Reprod. Biomed. Online. 2008; 16: 504-513Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 3Eberhard J. Ståhl O. Giwercman Y. Cwikiel M. Cavallin-Ståhl E. Lundin K.B. Flodgren P. Giwercman A. Impact of therapy and androgen receptor polymorphism on sperm concentration in men treated for testicular germ cell cancer: A longitudinal study.Hum. Reprod. 2004; 19: 1418-1425Crossref PubMed Scopus (21) Google Scholar, 4Tüttelmann F. Rajpert-De Meyts E. Nieschlag E. Simoni M. Gene polymorphisms and male infertility—A meta-analysis and literature review.Reprod. Biomed. Online. 2007; 15: 643-658Abstract Full Text PDF PubMed Scopus (183) Google Scholar). We previously discovered and validated a germ-cell-specific protein TEX101 1The abbreviations used are:TEX101testis-expressed protein 101LY6Klymphocyte antigen 6KACNacetonitrileADAM29A disintegrin and metalloproteinase domain-containing protein 29AGCautomatic gain controlCADDcombined annotation dependent depletionCFTRcystic fibrosis transmembrane conductance regulatorCIDcollision-induced dissociationDPEP3dipeptidase 3BH-adjusted t-testBenjamini–Hochberg-adjusted t-testFDRfalse discovery rateGPIglycosylphosphatidylinositolLEDlight-emitting diodeLFQlabel-free quantificationMWUMann Whitney unpaired t-testNHSN-hydroxysuccinimidePCRpolymerase chain reactionPRMparallel reaction monitoringSIFTsorting intolerant from tolerantSPseminal plasmaSNVSingle nucleotide variationSRMSelected reaction monitoringWTwild-type. 1The abbreviations used are:TEX101testis-expressed protein 101LY6Klymphocyte antigen 6KACNacetonitrileADAM29A disintegrin and metalloproteinase domain-containing protein 29AGCautomatic gain controlCADDcombined annotation dependent depletionCFTRcystic fibrosis transmembrane conductance regulatorCIDcollision-induced dissociationDPEP3dipeptidase 3BH-adjusted t-testBenjamini–Hochberg-adjusted t-testFDRfalse discovery rateGPIglycosylphosphatidylinositolLEDlight-emitting diodeLFQlabel-free quantificationMWUMann Whitney unpaired t-testNHSN-hydroxysuccinimidePCRpolymerase chain reactionPRMparallel reaction monitoringSIFTsorting intolerant from tolerantSPseminal plasmaSNVSingle nucleotide variationSRMSelected reaction monitoringWTwild-type. as a seminal plasma biomarker for the differential diagnosis of azoospermia and male infertility (5Drabovich A.P. Dimitromanolakis A. Saraon P. Soosaipillai A. Batruch I. Mullen B. Jarvi K. Diamandis E.P. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.Sci. Transl. Med. 2013; 5: 212ra160Crossref PubMed Scopus (115) Google Scholar, 6Drabovich 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, 7Korbakis D. Brinc D. Schiza C. Soosaipillai A. Jarvi K. Drabovich A.P. Diamandis E.P. Immunocapture-selected reaction monitoring screening facilitates the development of ELISA for the measurement of native TEX101 in biological fluids.Mol. Cell. Proteomics. 2015; 14: 1517-1526Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 8Korbakis D. Schiza C. Brinc D. Soosaipillai A. Karakosta T.D. Légaré C. Sullivan R. Mullen B. Jarvi K. Diamandis E.P. Drabovich A.P. Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.BMC Med. 2017; 15: 60Crossref PubMed Scopus (36) Google Scholar). The precise functional role of TEX101 is not known, but based on mouse models it was suggested as a testicular germ-cell-surface chaperone involved in the maturation of four cell-surface proteins from the ADAM family (9Fujihara Y. Tokuhiro K. Muro Y. Kondoh G. Araki Y. Ikawa M. Okabe M. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 8111-8116Crossref PubMed Scopus (106) Google Scholar, 10Li W. Guo X.J. Teng F. Hou X.J. Lv Z. Zhou S.Y. Bi Y. Wan H.F. Feng C.J. Yuan Y. Zhao X.Y. Wang L. Sha J.H. Zhou Q. Tex101 is essential for male fertility by affecting sperm migration into the oviduct in mice.J. Mol. Cell. Biol. 2013; 5: 345-347Crossref PubMed Scopus (22) Google Scholar). Tex101 knockout in mice resulted in male sterility but normal sperm concentration, morphology, and other phenotypical characteristics (9Fujihara Y. Tokuhiro K. Muro Y. Kondoh G. Araki Y. Ikawa M. Okabe M. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 8111-8116Crossref PubMed Scopus (106) Google Scholar). In the absence of TEX101 protein, ADAM 3–6 proteins were not properly processed and degraded. However, mouse data could not be translated into human studies because ADAM3, ADAM5, and ADAM6 genes are noncoding pseudogenes, while ADAM4 is not present in the human genome (11Cho C. Testicular and epididymal ADAMs: Expression and function during fertilization.Nat. Rev. Urol. 2012; 9: 550-560Crossref PubMed Scopus (62) Google Scholar). Lack of stable human male germ cell lines hinders identification of TEX101-associated proteins in humans. testis-expressed protein 101 lymphocyte antigen 6K acetonitrile A disintegrin and metalloproteinase domain-containing protein 29 automatic gain control combined annotation dependent depletion cystic fibrosis transmembrane conductance regulator collision-induced dissociation dipeptidase 3 Benjamini–Hochberg-adjusted t-test false discovery rate glycosylphosphatidylinositol light-emitting diode label-free quantification Mann Whitney unpaired t-test N-hydroxysuccinimide polymerase chain reaction parallel reaction monitoring sorting intolerant from tolerant seminal plasma Single nucleotide variation Selected reaction monitoring wild-type. testis-expressed protein 101 lymphocyte antigen 6K acetonitrile A disintegrin and metalloproteinase domain-containing protein 29 automatic gain control combined annotation dependent depletion cystic fibrosis transmembrane conductance regulator collision-induced dissociation dipeptidase 3 Benjamini–Hochberg-adjusted t-test false discovery rate glycosylphosphatidylinositol light-emitting diode label-free quantification Mann Whitney unpaired t-test N-hydroxysuccinimide polymerase chain reaction parallel reaction monitoring sorting intolerant from tolerant seminal plasma Single nucleotide variation Selected reaction monitoring wild-type. As an alternative, we suggested that the functional role of TEX101 could be studied in human clinical samples, such as spermatozoa. Our previous work on TEX101 levels in seminal plasma revealed a small population of men with high sperm count but very low levels of TEX101 protein in seminal plasma and spermatozoa (8Korbakis D. Schiza C. Brinc D. Soosaipillai A. Karakosta T.D. Légaré C. Sullivan R. Mullen B. Jarvi K. Diamandis E.P. Drabovich A.P. Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.BMC Med. 2017; 15: 60Crossref PubMed Scopus (36) Google Scholar). In this work, we hypothesized that some genomic alterations, such as natural knockouts or single nucleotide variations, could result in undetectable or low levels of TEX101 protein. We suggested that spermatozoa obtained from such men could be used as knockout or knockdown models to identify proteins degraded in the absence of TEX101 and discover the functional interactome of TEX101 in humans. Collectively, such data could support in humans the previously suggested function of TEX101 as a cell-surface chaperone (9Fujihara Y. Tokuhiro K. Muro Y. Kondoh G. Araki Y. Ikawa M. Okabe M. Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 8111-8116Crossref PubMed Scopus (106) Google Scholar). The objectives of this study were to identify potential genomic alterations that could impact levels of TEX101 protein and verify those levels experimentally in human spermatozoa samples. According to power calculations (one-tailed Fisher's exact test, a = 0.05 and 80% power), at least 25 men in each group (prevasectomy and unexplained infertility) would be required to detect an increase of rs35033974hh prevalence from 1.5% (prevalence in the general population) to 28.6% (hypothetical prevalence in men with unexplained infertility). The latter number was calculated as a ratio of rs35033974hh prevalence (1.5%) versus the prevalence of unexplained male infertility in the general population (70% of 7.5%) (12Kothandaraman N. Agarwal A. Abu-Elmagd M. Al-Qahtani M.H. Pathogenic landscape of idiopathic male infertility: New insight towards its regulatory networks.NPJ Genom. Med. 2016; 1: 16023Crossref PubMed Scopus (31) Google Scholar). Furthermore, we suggested that differential proteomic profiling of rs35033974hh spermatozoa could identify proteins degraded in the absence of TEX101. According to power calculations, differential profiling of spermatozoa of four wild-type (WT) and four rs35033974 homozygous men could identify proteins down-regulated at least 2.4-fold, assuming 80% power, α = 0.05, 1.8% coefficient of variation for log2-transformed LFQ intensity values, and a one-tailed t test (G*Power software, v3.1.7, Heinrich Heine University Dusseldorf). GraphPad Prism (v5.03) was used to generate scatter plots, perform statistical analysis, and calculate receiver operating characteristic area under the curves. Nonparametric Mann–Whitney U test was used to compare TEX101 levels in seminal plasma of WT and heterozygous men, and p values <0.05 were considered statistically significant. Semen samples (n = 386) were collected with informed consent from patients with the approval of the institutional review boards of Mount Sinai Hospital (approval #08–117-E) and University Health Network (#09-0830-AE). Samples were obtained from healthy fertile men before vasectomy and individuals diagnosed with oligospermia or unexplained infertility. Clinical parameters are summarized in Table I. The unexplained infertility group included men who were not able to father a pregnancy after one year of regular unprotected intercourse, with normal sperm concentration of greater than 15 million/ml. After liquefaction, semen samples were centrifuged three times at 13,000 g for 15 min at room temperature. Spermatozoa and SP were separated and stored at −80 °C. Samples were analyzed retrospectively. For the differential proteomic analysis, spermatozoa samples were obtained from four men homozygous for TEX101 c.296G>T variant (rs35033974hh), diagnosed with oligospermia (n = 2) and unexplained infertility (n = 2), median age of 29.5 years, sperm concentration 2–30 million/ml, and TEX101 concentration in SP 3.5–47 ng/ml. Spermatozoa obtained from the age-matched WT fertile men referred for vasectomy (n = 4, sperm concentration >15 million/ml and TEX101 concentration in SP of 8,000–12,500 ng/ml) were selected as a control group.Table IClinicopathological variables of 386 patientsClinical parametersN%Number of patients386100Median age [range]41 [19–63]Ethnic background of patientsAfrican-Canadian153.9Asian379.6European22658.5Hispanic61.5Indo-Canadian61.5Middle Eastern143.6Native Canadian51.3Unspecified/unavailable7720.1Diagnosis [range of sperm concentration, mln/ml]Fertile pre-vasectomy379.6Unexplained infertility (15–36 mln/ml)175 [15–36]45.3Oligospermia174 [0.1–15]45.1 Open table in a new tab Genomic DNA was extracted from spermatozoa using QIAamp DNA Mini Kit (Qiagen, Inc.). Spermatozoa were washed twice with phosphate-buffered saline (PBS). Cells were lysed in the presence of proteinase K and DNA bound to the membrane was washed and eluted. DNA purity and concentration were measured by spectrophotometer (NanoDrop 8000, Thermo Scientific). Forward (5′-ACAGGACTGAGACAGCCAT-3′) and reverse (5′-TCCAGGGTACCTGTGGTCTC-3′) primers were designed to amplify a 197 base pair fragment of TEX101 gene encompassing the rs35033974 polymorphism. Polymerase chain reaction (PCR) was performed with 50 ng of genomic DNA, 1.2 units of Phusion High-Fidelity DNA polymerase (Thermo Scientific) in Phusion HF Buffer, 200 μm deoxynucleoside triphosphates, and 0.5 μm primers using mastercycler thermal cycler (Eppendorf). PCR included an initial denaturation step at 98 °C for 1 min, followed by 40 cycles of denaturation at 98 °C for 10 s, annealing at 64 °C for 30 s and extension at 72 °C for 30 s, with a final extension at 72 °C for 7 min. PCR products were confirmed with 1.5% agarose gel electrophoresis and purified with QIAquick PCR Purification Kit (Qiagen). Sequencing of PCR products (n = 386 men) was performed by the Centre for Applied Genomic (Hospital for Sick Children, Toronto). Spermatozoa pellets were washed twice with PBS, lysed with 0.1% RapiGest SF (Waters, Milford, MA) in 50 mm ammonium bicarbonate, and sonicated three times for 30 s. Cell lysates were then centrifuged at 15,000 g for 15 min at 4 °C. Total protein in each spermatozoa or SP sample was measured by the bicinchoninic acid assay. Ten μg of total protein per patient sample in 50 mm ammonium bicarbonate were used for protein digestion. RapiGest SF 0.05% with 5 mm dithiothreitol at 65 °C for 30 min were used to denature proteins (purified recombinant human rhTEX101 and proteins from spermatozoa and SP) and reduce disulfide bonds. Free thiols were then alkylated with 10 mm iodoacetamide in the dark for 40 min at room temperature. Protein digestion was completed overnight at 37 °C in the presence of sequencing grade Glu-C enzyme obtained from Promega (1:20 Glu-C: total protein) and supplemented with 5% acetonitrile to enhance Glu-C activity. Digestion in the presence of ammonium bicarbonate at pH 7.8 ensured specific cleavage after glutamine residues. Trifluoroacetic acid (1%) was then used to inactivate Glu-C and cleave RapiGest SF detergent. Synthetic peptides representing the WT (AITIVQHSSPPGLIV*TSYSNYCE) and the G99V variant (AITIVQHSSPPVLIV*TSYSNYCE) forms of TEX101 were labeled with 13C5-, 15N-valine at the residue 102 and were used as internal standards spiked-in after digestion at final concentrations of 100 fmol/μl and 250 fmol/μl, respectively. Digests were desalted, and peptides were extracted by C18 OMIX tips (Varian, Inc., Lake Forest, CA). Peptides were eluted into 3 μl of 70% acetonitrile with 0.1% formic acid and analyzed by an EASY-nLC 1000 nanoLC coupled to Q ExactiveTM Plus Hybrid Quadrupole-OrbitrapTM Mass Spectrometer (Thermo Fischer Scientific). To evaluate Glu-C specificity and efficiency of digestion, rhTEX101 protein was digested and analyzed in the data-dependent discovery mode. Raw files were analyzed using the Proteome Discoverer™ software (Thermo Scientific, version 1.4.1.14), and specific generation of AITIVQHSSPPGLIVTSYSNYCE peptide (m/z = 1,268.6) was confirmed. Uniqueness of these peptides in the human proteome was confirmed by Basic Local Alignment Search Tool (http:/blast.ncbi.nlm.nih.gov/Blast.cgi). Following that, rhTEX101 and SP were digested with Glu-C and analyzed in the unscheduled targeted PRM mode. In the final optimized PRM method, heavy-isotope-labeled peptide internal standards and an additional endogenous TEX101 peptide TAILATKGCIPE (m/z = 637.3) were monitored (supplemental Table S1). A four-step 16-min gradient was used: 20% to 40% of buffer B for 8 min, 40% to 65% for 2 min, 65% to 100% for 2 min, and 100% for 4 min. PRM settings were the following: 3.0 eV in-source collision-induced dissociation (CID), 17,500 MS2 resolving power at 200 m/z, 3 × 106 automatic gain control (AGC) target, 100 ms injection time, 2.0 m/z isolation window, optimized collision energy at 27, and 100 ms scan times. Total TEX101 protein was enriched from SP and spermatozoa using an in-house anti-TEX101 mouse monoclonal antibody 34ED556. Briefly, protein G purified 34ED556 monoclonal antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow beads (GE Healthcare). Fifty μl of beads (∼25 μg of 34ED556) in 0.1% BSA were incubated overnight at 4 °C with seminal plasma or spermatozoa lysate. After binding, beads were washed three times with tris buffer saline (50 mm Tris, 150 mm NaCl, pH 7.5) followed by washing with 50 mm ammonium bicarbonate. Proteins were digested overnight on beads using Glu-C. Supernatants were acidified with 1% TFA. Heavy peptides (200 fmol of WT and 500 fmol of G99V) were spiked into each sample after digestion. Digests were desalted, and peptides were measured by PRM assay. Raw files were analyzed with Skyline software (v3.6.0.10493), and the relative abundances of WT or G99V variant TEX101 forms were calculated using the light-to-heavy peptide ratios. Spermatozoa pellets from eight men (four WT and four rs35033974hh) were lysed with 0.1% RapiGest SF in 50 mm ammonium bicarbonate. Cell lysates were centrifuged at 15,000 g for 15 min at 4 °C to remove debris, and total protein concentration was measured using BCA assay. Proteins (225 μg per sample) were denatured, reduced with 5 mm dithiothreitol, alkylated with 10 mm iodoacetamide, and digested overnight with trypsin (Sigma-Aldrich) at 37 °C. Off-line strong cation exchange chromatography fractionation was used to facilitate deep proteome analysis. Tryptic peptides were diluted with mobile phase A (0.26 m formic acid in 10% acetonitrile [ACN] at pH 2–3) and were loaded onto PolySULFOETHYL A™ column (2.1 mm inner diameter × 200 mm, 5 μm, 200 Å, The Nest Group, Inc., MA). Peptides were separated with a 60-min three-step HPLC gradient (Agilent 1100) and eluted at 200 μl/min with 1 m ammonium formate (0–15% for 5–25 min, 25% at 35 min, and 100% at 50 min). Twenty-seven 400 μl fractions were initially collected but then pooled into 13 fractions based on absorbance profiles. Peptides of each strong cation exchange chromatography fraction were concentrated with C18 OMIX tips and analyzed by an EASY-nLC 1000 system coupled to a Q ExactiveTM Plus mass spectrometer in technical duplicates for each fraction (13Begcevic I. Brinc D. Drabovich A.P. Batruch I. Diamandis E.P. Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas.Clin. Proteomics. 2016; 13: 11Crossref PubMed Scopus (45) Google Scholar, 14Cho C.K. Drabovich A.P. Karagiannis G.S. Martínez-Morillo E. Dason S. Dimitromanolakis A. Diamandis E.P. Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome.Clin. Proteomics. 2013; 10: 2Crossref PubMed Scopus (22) Google Scholar). Peptides were separated with a 15-cm C18 analytical column using a 90-min LC gradient at 300 nl/min flow rate. Full MS1 scans (400 to 1,500 m/z) were acquired with the Orbitrap analyzer at 70,000 full width at half maximum resolution in the data-dependent mode, followed by 12 data-dependent MS2 scans at 17,500 full width at half maximum. Only +2 and +3 charge states were subjected to MS2 fragmentation. XCalibur software (v. 2.0.6; Thermo Fisher Scientific) was utilized to generate raw files. For protein identification and label-free quantification, raw files were analyzed with MaxQuant software (version 1.5.2.8). MaxQuant searches were performed against the nonredundant Human UniprotKB/Swiss-Prot database (HUMAN5640_sProt-072016) at 1.0% FDR. Search parameters included: trypsin enzyme specificity, two missed cleavages, minimum peptide length of seven amino acids, minimum identification of one razor peptide, fixed modification of cysteines by carbamidomethylation, and variable modification of methionine oxidation and N-terminal protein acetylation. The mass tolerance was set to 20 ppm for precursor ions and 0.5 Da for fragment ions with top 12 MS/MS peaks per 100 Da. MaxLFQ algorithm facilitated label-free relative quantification of proteins (15Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2687) Google Scholar). ProteinGroups.txt file was uploaded to Perseus software (version 1.5.5.3) to facilitate statistical analysis (16Cox J. Mann M. 1D and 2D annotation enrichment: A statistical method integrating quantitative proteomics with complementary high-throughput data.BMC Bioinformatics. 2012; 16: S12Crossref Scopus (413) Google Scholar). Proteins classified as “only identified by site,” “reverse,” and “contaminants” were filtered out, and LFQ intensities were log2-transformed. Missing LFQ values were imputed with the down shift of 1.8 and distribution width of 0.45 to ensure normal distribution, and average LFQ intensities for two technical replicates were calculated. A two-sample t test with Benjamini–Hochberg FDR-adjusted p values was applied, and 5.0% FDR with calculated constant for variance correction s0 = 0.4 were used to select proteins differentially expressed in rs35033974hh men. Data were visualized with volcano plots. Significant up- or down-regulated proteins were filtered for the cell-surface and secreted proteins with the testicular-tissue-elevated (tissue-enriched, group enriched, and tissue-enhanced) expression according to the Human Protein Atlas, version 13 (1Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. Sivertsson Å. Kampf C. Sjöstedt E. Asplund A. Olsson I. Edlund K. Lundberg E. Navani S. Szigyarto C.A. Odeberg J. Djureinovic D. Takanen J.O. Hober S. Alm T. Edqvist P.H. Berling H. Tegel H. Mulder J. Rockberg J. Nilsson P. Schwenk J.M. Hamsten M. von Feilitzen K. Forsberg M. Persson L. Johansson F. Zwahlen M. von Heijne G. Nielsen J. Pontén F. Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (7240) Google Scholar). To quantify candidate proteins, we developed and applied Tier 2 SRM assays, as previously described (17Drabovich 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, 18Karakosta T.D. Soosaipillai A. Diamandis E.P. Batruch I. Drabovich A.P. Quantification of human kallikrein-related peptidases in biological fluids by multiplatform targeted mass spectrometry assays.Mol. Cell. Proteomics. 2016; 15: 2863-2876Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Drabovich 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, 20Martínez-Morillo E. Nielsen H.M. Batruch I. Drabovich A.P. Begcevic I. Lopez M.F. Minthon L. Bu G. Mattsson N. Portelius E. Hansson O. Diamandis E.P. Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.J. Proteome Res. 2014; 13: 1077-1087Crossref PubMed Scopus (43) Google Scholar, 21Drabovich A.P. Diamandis E.P. Combinatorial peptide libraries facilitate development of multiple reaction monitoring assays for low-abundance proteins.J. Proteome Res. 2010; 9: 1236-1245Crossref PubMed Scopus (51) Google Scholar). Briefly, LC-MS/MS peptide identification data were used to select proteotypic tryptic peptides and develop SRM assays. Choice of peptides was confirmed with the SRM Atlas (www.srmatlas.org). For each protein, peptides with 7–20 amino acids and without missed cleavages were chosen, and heavy-isotope-labeled peptide internal standards were synthesized. Several unscheduled 30-min SRM methods were prepared and run with a pool of spermatozoa digest with TSQ QuantivaTM triple quadrupole mass spectrometer (Thermo Scientific). The three most intense transitions were selected for each heavy or light peptide. Finally, 20 heavy and light peptides were scheduled within 2-min intervals during a 30-min gradient in a single multiplex SRM assay (supplemental Table S2). The parameters for SRM assay included: positive polarity, 150 V declustering and 10 V entrance potentials, 300°C ion transfer tube temperature, optimized collision energy values, 20 ms scan time, 0.4 Q1 and 0.7 Q3 full width at half maximum resolutions, and 1.5 mTorr Q2 argon pressure. Because one rs35033974 homozygote spermatozoa sample was fully consumed in the discovery experiment, candidate proteins were quantified in three rs35033974 homozygote and four WT spermatozoa samples. Spermatozoa lysates (10 μg protein) were digested by trypsin. TEX101 and DPEP3 internal standards with trypsin-cleavable tags" @default.
• W2900768108 created "2018-11-29" @default.
• W2900768108 creator A5046304922 @default.
• W2900768108 creator A5056642098 @default.
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• W2900768108 creator A5084226800 @default.
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• W2900768108 date "2019-02-01" @default.
• W2900768108 modified "2023-10-18" @default.
• W2900768108 title "Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974*" @default.
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