MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v05/MSV000091662/primaryOK2000000ProteomicsMartin M. MatzukOrbitrap EclipseMus Musculus (ncbitaxon:10090)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=c620d5983668403c89512f6574fc1a7emmatzuk@bcm.eduBaylor College of MedicineMassIVE102UNIMOD:27 - "Pyro-glu from E."UNIMOD:28 - "Pyro-glu from Q."UNIMOD:35 - "Oxidation or Hydroxylation."The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing. To this end, we used CRISPR-EZ to create mice lacking the entire coding region of Prss37, used pronuclear injection to create transgenic mice expressing human PRSS37, intercrossed these lines to generate humanized mice, and performed LC-MS/MS of KO and control tissues to identify proteomic perturbances that could attribute a molecular function to PRSS37. We found that our newly generated Prss37 KO mouse line is sterile, our human transgene rescues the sterility phenotype of KO mice, and our proteomics data not only yields novel insight into the proteome as it evolves along the male reproductive tract, but also demonstrates the proteins significantly influenced by PRSS37 loss. In summary, we report vast biological insight including insight into PRSS37 function and the generation of a novel tool for contraceptive evaluation.Molecular dissection and testing of PRSS37 function through LC-MS/MS and the generation of a PRSS37 humanized mouse model.Sutton Courtney C, Nozawa Kaori K, Kent Katarzyna K, Saltzman Alexander A, Leng Mei M, Nagarajan Sureshbabu S, Malovannaya Anna A, Ikawa Masahito M, Garcia Thomas X TX, Matzuk Martin M MMliquid chromatography tandem mass spectroscopy, Male, Product, Laboratory, CG 1618, multiple endocrine neoplasia syndrome, Mus domesticus, Addresses, dNSF, CASP-14, Progress Reports, neutral molecular compounds, salM, House Mouse, Sunday driver 1, prevention, X-linked achromatopsia incomplete, DmNSF, Summary Report, BCM, Pharmaceutical Product, dNSF1, dNsf1, Proteases, CG6464, Injectables, prevention and control, molecule, Summary Reports, male genitals, molecula, colorblindness, LCMSMS, molecules, Salm, Sperm surface protein, Man (Taxonomy), reference sample, Progress Report, Tissue, male genitalia, NOZC-1, Swiss Mice, Serine Proteinases, Molekuel, Boys, Protein highly expressed in testis, Proteinases, Sub-Fertility, preventive measures, Progress, blue-Mono-cone-monochromatic type, male genital organ, Field Reports, SalM, medicine, Pharmaceutical, ELL1, infertility, Ell1, JIP-4, slam, TG, house mouse, achromatopsia incomplete X-linked, LC-MS2, male genital system, Dissections, PHET, DmelCG6464, TNFRSF13A, Proteinase, preventive therapy, MBS85, Multiple endocrine neoplasms, DmelCG10120, Modern, LC-MS/MS, mouse, Contraceptives, Ids, cone dystrophy 5, X-chromosome-linked achromatopsia, familial endocrine, spalt, Sperm-specific protein, Investigative Report, humanised mouse, Mini-ICE, Pharmaceutic, multiple endocrine adenomatosis, SPALT, END, region, dnsf1, Tnfrsf13, Proliferation-inducing protein 6, Mus musculus, Serine, Caspase-14 subunit p10, function., Spalt, male organism reproductive system, mice, Swiss Mouse, Field, Adenomatosis, Caspase-14 subunit p19, MICE, human, domesticus, Phenotypes, Endocrine neoplasia, Report, Agents, multiple, Sperm-associated antigen 9, liquid chromatography-tandem mass spectroscopy, C19orf17, male genital tract, NOZAWA, anon-WO0118547.278, Mouse, NSF-1, CBBM, multiple endocrine neoplasia syndrome(s), l(2)03602, Mdh-NADP, Proteomes, humans, 3.4.22.-, male organism genitalia, MPS2, human being, X-linked, Peptidomics, color blindness blue mono cone monochromatic type, Comt, men syndromes, NSF1, Gene, mini-ICE, Multiple endocrine neoplasia, Protease, JNK-interacting protein 4, LC-MS-MS, NSF, Nsf, Human, Male Contraceptive, Subfertility, CG1618, Investigative, Homo sapiens, House, Injection, CT89, LC-MSMS, humanized hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr mutant mice, Gene Products, Mus musculus domesticus, Mice, Man, male reproductive tract, Tryx2, Drugs, Mitogen-activated protein kinase 8-interacting protein 4, study, SIDS, Swiss, Serine Proteinase, incomplete achromatopsia X-linked, JLP, systema genitale masculinum, multiple endocrine neoplasia, Injectable, men syndrome, drugs, Abstract, reproductive system of male organism, p85, p84, Reproductive Sterility, colour blindness blue mono cone monochromatic type, DmelCG1618, Serine Protein Hydrolases, site, TRYX2, Preparation, Investigative Reports, molecular function, Contraceptive, HHT1, Controlled, Pharmaceuticals, com, molecular function unknown, Reproductive, Products, Controlling, Human lung cancer oncogene 6 protein, Edg, BCMA, Multiple Endocrine Neoplasia, drug, MDH, Proteins, total expressed protein, function, ME, B1164, Cancer|testis antigen 89, LC/MS/MS, Mus, Protein, Research Reports, Familial endocrine adenomatosis, sequence, 3602, HLC-6, dNSF-1, Men, MEN, Me, CD269, JNK-associated leucine-zipper protein, SAL, Sal, Pharmaceutic Preparations, prophylaxis, LENG3, Protein Hydrolases, mem, House Mice, men, RGD1311823, Tnfrsf13a, 1700016G05Rik, primary structure of sequence macromolecule, Laboratory Mice, sal, Drug, Protein Gene Products, Gene Proteins, Preparations, PPP1R68, Reports, control, genitalia of male organism, Modern Man, Sterility, Male Contraceptives, liquid chromatography tandem mass spectrometry, Multiple endocrine adenomatosis, Serine Protease, Male Contraceptive Agents, ORW1, Pharmaceutical Products, Summary, Laboratory Mouse, salII, Field Report, CG10120, Pharmaceutical Preparationconcomitant conditions, liquid chromatography tandem mass spectroscopy, CRISPR Locus, CRISPR Loci, Clustered Regularly Interspaced Short Palindromic Repeat, Male, Product, multiple endocrine neoplasia syndrome, Laboratory, CRISPR Spacer, Mus domesticus, CASP-14, Progress Reports, neutral molecular compounds, Sunday driver 1, House Mouse, prevention, Recombinant Transgene, Biological, Summary Report, Pharmaceutical Product, Proteases, Biological Product, Injectables, prevention and control, Effect, molecule, Summary Reports, Arrays, male genitals, molecula, molecules, LCMSMS, Sperm surface protein, Man (Taxonomy), Biologic Drugs, reference sample, Progress Report, Elements, Transgenic Mouse, Natural, associated disease, Tissue, male genitalia, Serine Proteinases, Swiss Mice, Molekuel, Boys, Biological Drugs, Protein highly expressed in testis, Proteinases, Sub-Fertility, preventive measures, CRISPRs, Progress, male genital organ, Biological Medicine, Field Reports, medicine, Pharmaceutical, ELL1, infertility, Ell1, JIP-4, Medicine, sequelae, Medicines, house mouse, Biologic Drug, LC-MS2, male genital system, PHET, CRISPR Spacer Sequences, CRISPR Clusters, Proteinase, preventive therapy, Multiple endocrine neoplasms, DmelCG10120, Biologic Products, Founder, concomitant disease, Modern, mouse, LC-MS/MS, Contraceptives, CRISPR Arrays, CRISPR-Cas, Clusters, familial endocrine, CRISPR Element, Sperm-specific protein, Investigative Report, Biopharmaceuticals, Mini-ICE, Recombinant Transgenes, Biologic Product, Pharmaceutic, multiple endocrine adenomatosis, END, region, Proliferation-inducing protein 6, Mus musculus, sequels, Serine, Element, Caspase-14 subunit p10, male organism reproductive system, Biological Medicines, mice, Biological Drug, Swiss Mouse, Field, Adenomatosis, Transgenic Founder, Caspase-14 subunit p19, Biologics, Transgenic Founder Mice, MICE, human, domesticus, Phenotypes, Endocrine neoplasia, Report, Agents, multiple, Sperm-associated antigen 9, Cluster, C19orf17, liquid chromatography-tandem mass spectroscopy, Spacers, male genital tract, anon-WO0118547.278, Mouse, Spacer Sequences, multiple endocrine neoplasia syndrome(s), Mdh-NADP, Proteomes, humans, Founder Mice, 3.4.22.-, male organism genitalia, CRISPR-Cas Loci, coexistent disease, human being, Transgenic, Biologic Pharmaceuticals, Peptidomics, Effects, men syndromes, Gene, Multiple endocrine neoplasia, Protease, mini-ICE, Coding, CRISPR Array, JNK-interacting protein 4, LC-MS-MS, Human, Male Contraceptive, Subfertility, Investigative, Homo sapiens, House, coexistent conditions, Injection, CT89, LC-MSMS, Gene Products, Mus musculus domesticus, Mice, Medical, Man, male reproductive tract, CRISPR Spacers, Biologicals, Tryx2, Drugs, Mitogen-activated protein kinase 8-interacting protein 4, study, Clinical, Serine Proteinase, Swiss, Medical Coding, JLP, Natural Product, multiple endocrine neoplasia, systema genitale masculinum, CRISPR Spacer Sequence, CRISPR Sequences, CRISPR Cluster, CRISPR, Injectable, CRISPR-Cas Locus, men syndrome, drugs, reproductive system of male organism, Reproductive Sterility, Loci, Serine Protein Hydrolases, site, TRYX2, Preparation, Investigative Reports, molecular function, Contraceptive, HHT1, Biologic, Spacer Sequence, Controlled, Pharmaceuticals, molecular function unknown, Reproductive, Products, Contraceptive Effects, Controlling, CRISPR Sequence, Human lung cancer oncogene 6 protein, Edg, Biologic Medicines, Multiple Endocrine Neoplasia, drug, MDH, Transgenic Mice, Proteins, total expressed protein, function, ME, Cancer|testis antigen 89, Transgenes, Sequences, LC/MS/MS, Mus, Sequence, Transgene, Protein, Research Reports, Familial endocrine adenomatosis, CRISPR Elements, Biopharmaceutical, sequence, HLC-6, Spacer, Men, MEN, Me, JNK-associated leucine-zipper protein, Pharmaceutic Preparations, prophylaxis, Protein Hydrolases, mem, House Mice, men, associated conditions, RGD1311823, Locus, 1700016G05Rik, primary structure of sequence macromolecule, Contraceptive Effect., Laboratory Mice, Recombinant, Drug, Protein Gene Products, Gene Proteins, Preparations, PPP1R68, Reports, control, genitalia of male organism, Modern Man, Sterility, Male Contraceptives, Array, CRISPR Cas Loci, liquid chromatography tandem mass spectrometry, Natural Products, Multiple endocrine adenomatosis, Serine Protease, Male Contraceptive Agents, ORW1, Pharmaceutical Products, Summary, Laboratory Mouse, Field Report, CG10120, Pharmaceutical PreparationTryx2, liquid chromatography tandem mass spectroscopy, LC-MS2, Dissections, LC/MS/MS, LCMSMS, liquid chromatography-tandem mass spectroscopy, LC-MSMS, humanized hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr mutant mice, LC-MS/MS, liquid chromatography tandem mass spectrometry, TRYX2, function, RGD1311823, 1700016G05Rik, humanised mouse., LC-MS-MSTryx2, liquid chromatography tandem mass spectroscopy, LC-MS2, Dissections, LC/MS/MS, LCMSMS, liquid chromatography-tandem mass spectroscopy, LC-MSMS, humanized hCYP1A1_1A2_Cyp1a1/1a2(-/-)_Ahr mutant mice, LC-MS/MS, liquid chromatography tandem mass spectrometry, TRYX2, function, RGD1311823, 1700016G05Rik, humanised mouse., LC-MS-MS0falseMolecular dissection and testing of PRSS37 function through LC-MS/MS and the generation of a PRSS37 humanized mouse modelLC-MS/MS of Prss37 WT and KO mice male reproductive tract. Title: Molecular dissection and testing of PRSS37 function through LC-MS/MS and the generation of a PRSS37 humanized mouse model Authors: Courtney Sutton1,2, Kaori Nozawa1,2, Katarzyna Kent1,2, Alexander Saltzman3, Mei Leng3, Sureshbabu Nagarajan1,2, Anna Malovannaya3,4, Masahito Ikawa5,6, Thomas X. Garcia1,2,7, and Martin M. Matzuk1,2,* Email Addresses and ORCID IDs: Courtney Sutton: courtney.sutton@bcm.edu, 0000-0001-9994-990X Kaori Nozawa: nozawa.kaori.029@gmail.com, 0000-0002-2256-278X Katarzyna Kent: kkent@bcm.edu, 0000-0002-4528-3425 Alexander Saltzman: alexander.saltzman@bcm.edu, 0000-0003-4166-9555 Mei Leng: mleng@bcm.edu, 0000-0001-7231-8665 Sureshbabu Nagarajan: nsureshh@gmail.com, 0000-0003-3602-3709 Anna Malovannaya: malovann@bcm.edu, 0000-0003-2953-6485 Masahito Ikawa: ikawa@biken.osaka-u.ac.jp, 0000-0001-9859-6217 Thomas X. Garcia: thomas.garcia@bcm.edu, 0000-0001-7761-2660 Martin M. Matzuk: mmatzuk@bcm.edu, 0000-0002-1445-8632 *Correspondence to: Martin M. Matzuk, M.D., Ph.D. ABSTRACT The quest for a non-hormonal male contraceptive pill for men still exists. Serine protease 37 (PRSS37) is a sperm-specific protein that when ablated in mice renders them sterile. In this study we sought to examine the molecular sequelae of PRSS37 loss to better understand its molecular function, and to determine whether human PRSS37 could rescue the sterility phenotype of knockout (KO) mice, allowing for a more appropriate model for drug molecule testing. To this end, we used CRISPR-EZ to create mice lacking the entire coding region of Prss37, used pronuclear injection to create transgenic mice expressing human PRSS37, intercrossed these lines to generate humanized mice, and performed LC-MS/MS of KO and control tissues to identify proteomic perturbances that could attribute a molecular function to PRSS37. We found that our newly generated Prss37 KO mouse line is sterile, our human transgene rescues the sterility phenotype of KO mice, and our proteomics data not only yields novel insight into the proteome as it evolves along the male reproductive tract, but also demonstrates the proteins significantly influenced by PRSS37 loss. In summary, we report vast biological insight including insight into PRSS37 function and the generation of a novel tool for contraceptive evaluation.Fri Apr 07 15:03:00 BST 2023MSV00009166237452050