{"database":"MassIVE","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://massive-ftp.ucsd.edu/v03/MSV000087493/"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":0,"searchCount":0},"additional":{"submitter":["Laurence Florens"],"full_dataset_link":["https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=cefffaedf3f5475faa974d35f1de0e56"],"submitter_email":["laf@stowers.org"],"sample_protocol":[""],"repository":["MassIVE"],"file_size":["102"],"ptm_modification":["MOD:01060 - \"A protein modification that effectively converts an L-cysteine residue to S-carboxamidomethyl-L-cysteine.\"","MOD:00719 - \"A protein modification that oxygenates an L-methionine residue to one of the diastereomeric L-methionine sulfoxide residues.\""],"data_protocol":[""],"omics_type":["Proteomics"],"instrument_platform":["LTQ"],"species":["Schizosaccharomyces Pombe (ncbitaxon:4896)"],"submitter_affiliation":["The Stowers Institute for Medical Research"],"pubmed_abstract":["Telomerase reverse transcriptase (TERT) and the noncoding telomerase RNA (TR) subunit constitute the core of telomerase. Additional subunits are required for ribonucleoprotein complex assembly and in some cases remain stably associated with the active holoenzyme. Pof8, a member of the LARP7 protein family is such a constitutive component of telomerase in fission yeast. Using affinity purification of Pof8, we have identified two previously uncharacterized proteins that form a complex with Pof8 and participate in telomerase biogenesis. Both proteins participate in ribonucleoprotein complex assembly and are required for wildtype telomerase activity and telomere length maintenance. One factor we named Thc1 (Telomerase Holoenzyme Component 1) shares structural similarity with the nuclear cap binding complex and the poly-adenosine ribonuclease (PARN), the other is the ortholog of the methyl phosphate capping enzyme (Bin3/MePCE) in metazoans and was named Bmc1 (Bin3/MePCE 1) to reflect its evolutionary roots. Thc1 and Bmc1 function together with Pof8 in recognizing correctly folded telomerase RNA and promoting the recruitment of the Lsm2-8 complex and the catalytic subunit to assemble functional telomerase."],"pubmed_title":["A putative cap binding protein and the methyl phosphate capping enzyme Bin3/MePCE function in telomerase biogenesis."],"pubmed_authors":["Páez-Moscoso Diego J DJ, Ho David V DV, Pan Lili L, Hildebrand Katie K, Jensen Kristi L KL, Levy Michaella J MJ, Florens Laurence L, Baumann Peter P"],"pubmed_title_synonyms":["SPI3, vp165, SMC3, Biocatalysts, Smc3, PLAP, l(3)L4740, Gene, CG4795, protein, protein-containing complex, Capt, DmelCG9802, protein polypeptide chains, dSMC3, c-Cbl-associated protein, polypeptide chain, capping protein, Telomerase Catalytic, Dcap, SH3 domain protein 5, Gene Products, mKIAA1296, l(3)S097074, cg9748, BIN3, Bin3, protein aggregate, DmelCG8276, IRAP, RHL3, D5Wsu46e, 2310065E01Rik, HACD1, CAP1-PEN, DFNB91, PI-6, R85FL, CG5074, Subunit, punc, ligand, Placental thrombin inhibitor, l(2)SH2 1014, MSTP057, proteins, Mmip1, PI6, BMH, MCAP, DmelCG9748, DmSMC3, Reverse Transcriptase, Enzyme, ROOT HAIRLESS 3, L3, BEL, Bel, Catalytic Subunit, Telomerase Reverse Transcriptase Catalytic Subunit, topoisomerase 6 subunit B, ms(3)Pneo85A, 4732490P18Rik, CG3451, CSPG6, Smcd, SH3P12, Telomerase Reverse Transcriptase, DmRH6, 2.1.1.-, protein complex, Proteins, CG9802, acu, Telomerase Reverse, SORB1, anon-85Ab, cyclase-associated protein, l(3)S047110, HYP6, MePCE, function, ms(3)neo30, Ponsin, Cytoplasmic antiproteinase, Telomerase, cpn, BRASSINOSTEROID INSENSITIVE 3, DmelCG4795, native protein, natural protein, CG5061, 9530001P15Rik, Protein, CAP, Cap, Ag 72H5, TCP2, Peptidase inhibitor 6, BG02184, l(2)SH1014, Mab77H5, PTI, 1700015F07Rik, CG33979, l(3)85Ac, CAP/Vinexin, CG9748, Bin3 homolog, cap, 77H5, DmelCG33979, DmSMC3/Cap, BcDNA:LD24380, Rexin, FLAF2, BCDIN3, SH3D5, Protein Gene Products, HUNKI, Gene Proteins, CG18409, CG18408, CG8276, Bcdin3, Transcriptase, Telomerase Catalytic Subunit., Sh3d5, Acap, BAM, Bicoid-interacting protein 3 homolog, l(3)L3, Biocatalyst, DCAP, SMC3L1, DmelCG18408, HUNK1, l(2)06955, ELONGATED HYPOCOTYL 6, rexin, gp160, Ag72H5, 2010309L07Rik, ATTOP6B"],"description_synonyms":["sodium salt, l(4)16, Antigenic Determinants, Dinitrilotetraacetate, ammonium formate, Ethylenedinitrilotetraacetic Acid, 13C-labeled, cadmium salt, Potassium EDTA, calm1, Keratin-Associated Proteins, SDH, chronic obstructive pulmonary disease (COPD), Basodexan, magnesium formate, Apaf-1, mg/ml, zinc salt, prevention, Dicobalt, dmTAF[[II]]230, 2-Propanone, Polypeptides, SDS, protein polypeptide chains, epitope function, hydrogen chloride, Edetic, T22F8_160, cobalt(II) formate dihydrate, Protease Inhibitor, GRP1, ATGPR7, Grp1, Chronic irreversible airway obstruction, 1, 2, GRP8, GRP7, Cal49A, WMS, 3-Propanetriol, Disodium Salt, Oelsuess, TFIID TAF250, hac-1, antigenic determinant, cel, Glycine, DmelCG13176, N, PTPSTEP, AI327027, (24)NaCl, ORF19, CAFL - Chronic airflow limitation, CaCl2, proteins, Edetate, D17Mit170, Keratin Associated Proteins, AI047805, salt, Wasserstoffchlorid, Peptidohydrolase Inhibitor, C1 4B, Propanon, disease (COPD), autoimmune disease of retina, Rotations, 2-Ethanediylbis(N-(carboxymethyl)glycine), RED, Red, autolysin activity, Gallium EDTA, GPH, Calcium chloride anhydrous, nickel salt, SGS, Glyceritol, DmCalm, Epitope, dTAF[[II]]230, chronic obstructive lung disease [Ambiguous], dCaM, preventive therapy, cobalt (+2) salt, CaM KII, Trypsin/K, Dipotassium Salt, anon-53Fa, Keratin, TAF200, l(2)SH0173, Tl3, Tl2, ug/ml, HMG-CoAR, CSA2, CaM II, ACMICD, CHRONIC OBSTRUCTIVE AIRWAY DIS, common salt, sodium (4:1:1) salt, Pyroacetic ether, Dm1, magnesium salt, [4)-3, Hydrolase Inhibitor, Sepharose C1, Chlorwasserstoff, Magnesium Salt, 1110049F14Rik, Chromium EDTA, Airflow Obstruction, CG6829, Antigenic Determinant, dark/hac-1/dapaf-1, GLYCINE-RICH PROTEIN 8, Clinorotations, Chronic Obstructive Airways Disease, Acid, Chronic obstructive lung disease, CAMKIId, Gro, glycerine, Antagonists, CG11628, formate, Shwachman-Diamond type metaphyseal dysplasia, pooled, cromium (+3), Striatum-enriched protein-tyrosine phosphatase, Shwachman syndrome, N'-1, beta Trypsin, human, Sodium Salt, F2G1.4, Taf250, OBSTRUCTIVE PULMONARY DISEASE (COPD), bacteriolytic toxin activity, ammonium (4:1) salt, Edetate Disodium Calcium, Endopeptidase, Peptide Peptidohydrolase Inhibitor, chlorane, Disodium Versenate, lead (+2) salt, TAF230, humans, Ethylenediaminetetraacetic Acid, Gallium, F10B6_15, GRP1/cytohesin 1, nickel formate dihydrate, lead formate, chlorure d'hydrogene, Endopeptidase Inhibitors, Biocatalysts, aluminum salt, CAO - Chronic airflow obstruction, Ethylenediaminetetraacetic, Carbamide, CHRONIC OBSTRUCTIVE PULM DIS, Antigenic Specificity, Chronic obstructive pulmonary disease NOS, protein-containing complex, CG7826, F10B6.15, and RNA binding 1, Human, CG11633, Chronic obstructive pulmonary disease finding, glycyl alcohol, propanone, Determinant, PULMONARY DISEASE (COPD), Magnesium Disodium, AIR, dimethylcetone, Gene Products, CG7835, CG42273, CaM KMT, Peptide Hydrolase, Carmol, Man, dTAF[[II]]250, FAM39E, Hydrogen chloride, thallium (+1) salt, cell, (22)Na, Glycerin, Monosodium Salt, beta-Trypsin, rubidium salt, 4733401P19Rik, D-CaM, methanoic acid, Antagonist, Solution, dTAF250, Tetracemate, Enzyme, glycerolum, Protease Antagonists, ATGRP7, ATGRP8, Stannous EDTA, congenital lipomatosis of pancreas, NEC, Kochsalz, Peptide Peptidohydrolase Inhibitors, Chronic, peptidos, PHKD, Associated Protein, Dimethyl ketone, AI256814, Potassium, dark/dapaf-1/hac-1, cou, chronic obstructive airways disease NOS, dApaf-1, Proteins, COAD, Stannous, stepk, Coprin, BG:DS00004.13, chloridohydrogen, potassium formate, copper (+2) salt, COLD - Chronic obstructive lung disease, buffer, Copper, Smp1, Cell, chronic, dTAF230, Versene, polypeptide, Wash2, APAF1, Wash1, Lr, Temperatures, native protein, Natriumchlorid, carbonyldiamide, Hac-1, cupric formate, Strain, TAF[[II]]250/230, MFS1, MIXL, (COPD), chronic airway obstruction, NaCl, Edathamil, chronic obstructive, aluminum formate, CAL - Chronic airflow limitation, CG18069, Taf[[II]]250, AU020952, Calcium, lithium formate, holin, prophylaxis, Ethylene Dinitrilotetraacetate, CamKIIalpha, GR-RBP7, Specificity, GR-RBP8, SWDS, Dark/Dapaf-1/HAC1, Monopotassium Salt, Shwachman-Bodian-Diamond syndrome, Peptidohydrolase Inhibitors, REDK, Magnesium Disodium EDTA, DmelCG11628, ME-IV, Gene Proteins, Peptidase, Antiprotease, control, Disodium Ethylene Dinitrilotetraacetate, Pancreatic insufficiency and bone marrow dysfunction, chronic obstructive lung disease (disorder), Bra, Peptide Hydrolase Inhibitors, CCM1, hydrochloric acid, anon-EST:Posey59, Calcium Salt, chronic obstructive pulmonary disease, Ca2+/calmodulin-dependent protein kinase II, Hac-1/Dark, Endopeptidase Inhibitor, camKII, T22F8.160, CALML2, and rna binding 2, DD132, Sepharose C1 4B, Chronic Airflow Obstructions, DmelCG6829, classic hairy cell leukaemia, Peptide Hydrolase Inhibitor, zinc formate, lead salt, Ethylenedinitrilotetraacetic, Hydrogenchlorid, protein, Calcium Disodium, peptide, halite, ammonium tetraformate, peptido, Edetic Acid, congenital, chronic obstructive airways disease NOS (disorder), Keratin Associated, Inhibitors, Min, l(4)ar, Apaf1, Chronic Obstructive Pulmonary Disease (COPD), Hydrolase Inhibitors, protein aggregate, prevention and control, ARK, methyl ketone, Calcium Tetacine, MIX, DmelCG42273, DmelCG18069, Sepharose, Edetates, me75, CDPK1, COLD (chronic obstructive lung disease), Man (Taxonomy), reference sample, peptides, enzymes, NEC in ICD9CM_2006, E927b, min, arc, mAPC, copper, ark, free, l(4)102EFb, T1, Calcitetracemate, lipomatosis of pancreas, Chronic airflow limitation, alpha-Keratin, Calm, preventive measures, cloruro sodico, Calcium Disodium Versenate, CAMI, lithium salt, Disodium Calcitetracemate, SOLO DANCERS, GLYCINE RICH PROTEIN 7, CAMC, l(2)k08110, CaM kinase, C2orf34, Tetacine, Shwachman-Bodian syndrome, Strains, necrosis, dApaf1, glycine-rich RNA-binding protein 8, GPHYSD2, CHRONIC OBSTRUCTIVE PULMONARY DISEASE, ammonium (2:1) salt, epitope role, CPVT4, Cam1, CHRONIC, rock salt, obstructive lung disease, Calcium Disodium Edetate, Peptidase Inhibitor, D-Apaf-1, eluted material, 4B, dimethylketone, Modern, CAMKII, Dmel_CG7826, AI836084, TAFII-250, classic hairy cell leukemia, TAF250/230, Peptide Peptidohydrolase, Airflow Obstructions, Aceton, ur, pulmonary disease (COPD), Versenate, carbamide, TAFII250, CHRONIC OBSTRUCTIVE, glycerol, Distannous EDTA, 3.1.3.48, chronic obstructive pulmonary disease and allied conditions, Chronic airway disease, bacteriocin activity, NOS, WASH, nickel (+2) salt, Copper EDTA, Dmel_CG7835, COPD NOS, CLNMT, Mnb, MNB, CHRONIC OBSTRUCTIVE LUNG DIS, MILD1, Chronic airway obstruction, ammonium salt, Distannous, cold, Step, HCl, cobaltous formate, POF8, common, hac1, MASS, AW124434, CG17603, natrii chloridum, TAF[[II]], beta-Ketopropane, alpha Keratin, dapaf-1S, Clinorotation, Chronic Airflow Obstruction, H2NC(O)NH2, apaf-1, lysin activity, Peptidase Inhibitors, dapaf-1L, STEP, SR3-5, COPD - Chronic obstructive pulmonary disease, copper salt, Antiproteases, Sepharose 4B, chronic obstructive airways disease, Biocatalyst, Polypeptide, 2-ethanediylbis(N-(carboxymethyl))-, obstructive pulmonary disease (COPD), 3909, Strains and Sprains, Inhibitor, chronic obstructive lung disease, COLD, 1728, Cytokeratin, protein levels, CaMK II, d230, human being, Dapaf-1/HAC-1, DISEASE (COPD), cesium salt, FBN, Gene, dTAFII250, Protease, CAMIII, circadian rhythm, EfW1, Buffer, formic acid, Potassium Salt, Dark/Hac-1/dApaf1, cytohesin/GRP1, Karbamid, Glycerine, Cold, Hac1, potassium salt, Propanetriol, Dark/Hac-1/dApaf-1, Pulmonary Disease, Homo sapiens, polypeptide chain, dmTAF1, Taf230, ECTOL1, p63, 14C-labeled, Trihydroxypropane, p65, Harnstoff, AL024000, KMT, Protease Antagonist, l(2)03909, Low, Keratin Associated Protein, Sprains, cloruro de hidrogeno, TAF250, CamKII, hYAK3-2, Taf200, Cold Temperatures, l(2)3909, Ethylene, dapaf-1, chlorure de sodium, l(2)SH2 0323, Antigenic, Taf1p, dapaf, dark, calcium formate, Chronic Obstructive, calcium chloride anhydrous, OCTD, Sodium Chloride, Camkii, Chromium Salt, Neural-specific protein-tyrosine phosphatase, DARK, EDTA, CG8472, Agarose, cromium (+3) salt, Azeton, TAF, dArk, Dapaf-1, Dicobalt EDTA, 6-An-alpha-L-Galp-(1->3)-beta-D-Galp-(1->]n, Disodium EDTA, sodium formate, TCEP, Controlled, COPD, DYRK5, Gene., Controlling, DYRK1, COAD - Chronic obstructive airways disease, Dimethylketon, TAF[[II]]250, nickel formate, protein complex, Glyzerin, not elsewhere classified, CaM, Sprain, CAMKIIalpha, strontium formate, l(3)84Ab, apaf1, enzyme activity, strontium salt, Peptide, PULM DIS CHRONIC OBSTRUCTIVE, dCaMKII, Chronic obstructive pulmonary disease finding (finding), [HCl], calcium/calmodulin-dependent protein kinase II, natural protein, AI461935, p230, DCK, Protein, Cam, caM, CAM, Calcium/calmodulin-dependent protein kinase, Cal, Hydrochloride, TFIID, table salt, 3200001F09Rik, Dyrk1, GLYCINE-RICH RNA-BINDING PROTEIN 7, Dark, Determinants, l(2)SH0323, Disodium Ethylene, Dark/Apaf-I, WMS2, Data Base, CG13176, DmelCG8472, CYH1, chronic obstructive airway disease, 3-Trihydroxypropane, Temperature, TAF[[II]]230, PRSS, Chelaton 3, uree, Dops, cam, Dihydrate, CC1, Sodium chloride (NaCl), Tripcellim, TAF[II]250, CaMK-II, Disodium, CGI-97, dApaf-1/DARK/HAC-1, chromic formate, retina autoimmune disease, Protein Gene Products, Trypure, DmelCG17603, concentration, Modern Man, SSKS, [CaCl2], calcium salt, Peptid, cold (chronic obstructive lung disease), Chronic Obstructive Lung Disease, Chromium, dAPAF-1, TAF1"],"name_synonyms":["Protein Gene Products, Gene Products, Proteins, POF8., Gene Proteins, Gene, assay, determination, chemical analysis, Protein"],"pubmed_abstract_synonyms":["HERV-K(III) Pol protein, Networks, Forms, Kinship, Fission, metazoans, Telomere Length, protein, Schizosaccharomyces pombe, telomerase RNA, hTRT, RNase, Integrase, rat, protein polypeptide chains, Fission Yeast, neutrophilic leucocyte, Telomerase Catalytic, neutrophil leukocyte, hEST2, DKCB4, 1, Life Cycle, BIN3, Bin3, protein aggregate, HERV-K_19p13.11 provirus ancestral Pol protein, hTR RNA, PIP7S, fission yeast, RHL3, SCNX, Family Life Cycle, Lengthening, NCBP-NIP1 complex, Animalia, D5Wsu46e, RT, imd2, TERC, Kinship Network, Length Maintenance, HERV-K_1q23.3 provirus ancestral Pol protein, Subunit, DKCA2, THC, l(2)SH2 1014, Adenocor, proteins, purification, telomerase RNA component, bTR, isolation and purification, Nucleases, HERV-K_11q22.1 provirus ancestral Pol protein, beta-D-adenosine, ROOT HAIRLESS 3, Telomerase Reverse Transcriptase Catalytic Subunit, polymorphonuclear leukocyte, polynuclear neutrophilic leucocyte, topoisomerase 6 subunit B, ribonucleoprotein complex assembly, thc, RNA-Directed, Family Life Cycles, TR, 4-diol, telomerase RNA reverse transcriptase activity, 3R, Adenosin, RGD1565449, (2R, HERV-K_5q33.3 provirus ancestral Pol protein, Ado, WASP, wasp, mouse, Telomerase Reverse, HYP6, ALAZS, thrombocytopenia 1, S pombe, Telomerase, 4S, BRASSINOSTEROID INSENSITIVE 3, HERV-K107 Pol protein, neutrocyte, 9-beta-D-Ribofuranosidoadenine, RNP complex assembly, protein-RNA complex assembly, xparn, Maintenance, HERV-K_19q11 provirus ancestral Pol protein, IMD2, dLARP7, catalyst, TCP2, Filiation, l(2)SH1014, Acid, Yeast, HERV-K_3q27.2 provirus ancestral Pol protein, TCS1, parn-A, CG42569, Ribonuclease, Telomere, HERV-K110 Pol protein, POF8, adenine riboside, 6-Amino-9-beta-D-ribofuranosyl-9H-purine, BCDIN3, human, Life Cycles, RNA Directed DNA Polymerase, Reverse transcriptase, 9-beta-D-ribofuranosyl-9H-purin-6-amine, Alkaline Ribonuclease, HERV-K(C19) Pol protein, Bcdin3, RNA Directed, Biocatalyst, RNA-Dependent, Adenoscan, HERV-K_8p23.1 provirus ancestral Pol protein, HERV-K(HML-2.HOM) Pol protein, HERV-K_1q22 provirus ancestral Pol protein, ATTOP6B, polynuclear neutrophilic leukocyte, HERV-K10 Pol protein, Family Member, X-linked, Ribonuclease H, Biocatalysts, Gene, Network, Schizosaccharomyces malidevorans, protein-containing complex, Telomere Lengthening, animals, CG12058, X-linked recessive, X-linked thrombocytopenia with normal platelets, 5R)-2-(6-aminopurin-9-yl)-5-(hydroxymethyl)oxolane-3, HERV-K115 Pol protein, IN, hterc, polypeptide chain, Deoxynucleotide-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), isolation, RNA Dependent DNA Polymerase, Gene Products, HERV-K18 Pol protein, TP2, Alkaline, DmelCG8276, template, 1200003I18Rik, bovine telomerase RNA, Schizosaccharomyces pombeP, HERV-K(C1a) Pol protein, Research, neutrophil leucocyte, Revertase, neutrophilic leukocyte, TERC RNA, Reverse Transcriptase, Enzyme, Catalytic Subunit, CMM9, HERV-K_7p22.1 provirus ancestral Pol protein, Telomerase Catalytic Subunit, Holoenzyme, 9-beta-D-Ribofuranosyl-9H-purin-6-amine, D3Wsu161e, DAN, Kinship Networks, Family, Adenocard, RNase H, 9-beta-D-ribofuranosyladenine, X-linked thrombocytopenia, Telomerase Reverse Transcriptase, RNA, intermittent, Family Research, 2.1.1.-, protein complex, Proteins, thc1, MePCE, TRT, function, Ade-Rib, HERV-K102 Pol protein, Family Members, HERV-K108 Pol protein, RNA-protein complex assembly, native protein, natural protein, EST2, Protein, Acid Ribonuclease, core, DNA Polymerase, 3.1.26.4, RNA-Dependent DNA Polymerase, thrombocytopenia, CBC, 1700015F07Rik, Homeostasis, snRNA cap binding complex, Bin3 homolog, telomerase, Dmel_CG12058, HERV-K(C7) Pol protein, HERV-K113 Pol protein, C330027G06Rik, Protein Gene Products, XLT, multicellular animals, Gene Proteins, Telomere Length Maintenance, Fission Yeasts, THC1, DmelCG42569, PFBMFT1, CG8276, Transcriptase, Families, Telomerase Catalytic Subunit., Bicoid-interacting protein 3 homolog, PMN, mRNA cap binding complex, polymorphonuclear leucocyte, RNA Nucleases, poly, 2.7.7.49, Relatives, ELONGATED HYPOCOTYL 6, polymorphonuclear neutrophil, RNA Transcriptase, thrombocytopenia type 1"],"citation_count":["0"],"additional_accession":["PXD026230"]},"is_claimable":false,"name":"MudPIT analysis of S. pombe proteins interacting with Pof8","description":"To identify proteins interacting with Pof8, cell-free extracts of strains FP1547 harboring 3xFLAG epitope-tagged Pof8 and untagged control (FP1546) were diluted to 5 mg/ml in 8 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol) plus complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM PMSF, 1 mM EDTA, and 0.1 mM DTT and incubated with 500 ul of EZview Red Flag-agarose 50% slurry equilibrated in lysis buffer. The mix was incubated for 8 hours at 4 C with gentle rotation. Agarose beads were collected by centrifugation at 300 x g for 5 min with low brake and washed 5 times for 5 minutes with 5 ml of wash buffer (50 mM Tris-HCl pH 7.5, 250 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol) at 4 C with gentle rotation. Proteins were eluted from beads by incubation for 30 min with 500 ul elution buffer (50 mM Tris- HCl pH 7.5, 250 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol, and 100 ug/ml 3xFLAG peptide) at 4 C. The elution step was repeated two more times and the eluates were pooled. A 4 ul aliquot of the first eluate was used for Silver stained SDS PAGE. The elutes were treated with 0.1U of benzonase for 30 min at 37 C and further diluted with the same volume of 100 mM Tris-HCl, pH 8.5. The samples were then split in three 400ul aliquots, mixed with 100 ul of 100% TCA and incubated at 4 C overnight. After centrifugation at 14,000 rpm for 30 min at 4 C, the pellets were washed twice with 500 ul cold acetone and centrifuged at 14,000 rpm for 10 min after each wash. Air dried pellets were resuspended in 30 ul 100 mM Tris-HCl, pH 8.5, 8M Urea and pooled. 4.5 ul of 0.1M TCEP was added to the pooled solution to a final concentration of 5 mM and incubated for 30 min at room temperature. After the incubation, 1.8 ul of 0.5 M CAM was added and incubated for 30 min at RT in the dark. Proteins were then digested with 0.1 ug/ul Lys-C at 37 C overnight. The samples were then diluted to 2M Urea with 100 mM Tris-HCl, pH8.5, added with CaCl2 to 2 mM and digested with 0.1 ug/ul Trypsin at 37 C overnight. Finally, 90% formic acid was added to a final concentration of 5%. and samples were analyzed by MudPIT. Tandem mass (MS/MS) spectra were interpreted using ProLuCID against the NCBI database of S. pombe proteins supplemented with 177 sequences from common contaminants (human keratins, IgGs, proteolytic enzymes). To estimate relative protein levels, (dNSAFs) were calculated for each detected protein. dNSAF values take into account peptides shared between multiple proteins and the length of the protein for normalization.","dates":{"publication":"Mon May 24 12:07:00 BST 2021"},"accession":"MSV000087493","cross_references":{"pubmed":["35217638"]}}