Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/feature_alignment_OpenSWATHresult.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/SampleInformationList.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/PRPF8_E1311141148.prot.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_003_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_002_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_001_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_021_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_020_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_016_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_015_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_015_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_016_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_001_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_017_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_016_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_002_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_015_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_003_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_017_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_015_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_017_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_018_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_018_SW.wiff.scanftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_017_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_016_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_020_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_K140110_021_SW.wiffftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/PRPF8_InterProphet.pep.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_012.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120904_001.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/yanliu_L120831_013.mzXMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/08/PXD003278/PRPF8_forOpenSWATH.TraMLprimaryOK200003250aebersold@imsb.biol.ethz.chYansheng LiuMass SpectrometrySWATH MSNot availableAlternative splicingSwathProteomePrpf8http://www.ebi.ac.uk/pride/archive/projects/PXD003278Cell CultureProtein extraction and in-solution digestion. The cell pellets from three independent depletion experiments (control siRNA and PRPF8 depleted) were lysed on ice by using a lysis buffer containing 8 M urea (EuroBio), 40 mM Tris-base (Sigma-Aldrich), 10 mM DTT (AppliChem) and complete protease inhibitor cocktail (Roche). The resulted mixture was sonicated at 4 °C for 5 mins using a VialTweeter device (Hielscher-Ultrasound Technology) and centrifuged at 21130 g, 4 °C for 1 hr to remove the insoluble material. The supernatant protein mixtures were transferred and protein amount was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). Aliquots of 1 mg protein mixtures were reduced by 5 mM tris(carboxyethyl)phosphine (Sigma-Aldrich) and alkylated by 30 mM iodoacetamide (Sigma-Aldrich). Then 5 volumes of precooled precipitation solution containing 50% acetone, 50% ethanol, and 0.1% acetic acid was added to the protein mixture and kept at −20 °C overnight. The mixture was centrifuged at 20,400 g for 40 min. The pellets were washed with 100% acetone and 70% ethanol with centrifugation at 20,400 g for 40 min. The samples were then resolved by 100 mM NH4HCO3 and were digested with sequencing-grade porcine trypsin (Promega) at a protease/protein ratio of 1:50 overnight at 37 °C57. Digests were purified with Vydac C18 Silica MicroSpin columns (The Nest Group Inc.). Peptide amount was determined using Nanodrop ND-1000 (Thermo Scientific) and about 0.7 μg peptide mixtures were analyzed in each LC-MS run. An aliquot of retention time calibration peptides from iRT-Kit (Biognosys) was spiked into each sample before all LC-MS analysis at a ratio of 1:30 (v/v) to correct relative retention times between runs58. Shotgun measurement. The peptides digested from Cal51 lysate were all measured on an AB Sciex 5600 TripleTOF mass spectrometer operated in DDA mode. The mass spectrometer was interfaced with an Eksigent NanoLC Ultra 2D Plus HPLC system as previously described5,6,59. Peptides were directly injected onto a 20-cm PicoFrit emitter (New Objective, self-packed to 20 cm with Magic C18 AQ 3-μm 200-Å material), and then separated using a 120-min gradient from 2–35% (buffer A 0.1% (v/v) formic acid, 2% (v/v) acetonitrile, buffer B 0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 300 nL/min. MS1 spectra were collected in the range 360–1,460 m/z. The 20 most intense precursors with charge state 2–5 which exceeded 250 counts per second were selected for fragmentation, and MS2 spectra were collected in the range 50–2,000 m/z for 100 ms. The precursor ions were dynamically excluded from reselection for 20 s. SWATH-MS measurement. The same LC-MS/MS systems used for DDA measurements was also used for SWATH analysis 5,6,59. Specifically, in the present SWATH-MS mode, the SCIEX 5600 plus TripleTOF instrument was specifically tuned to optimize the quadrupole settings for the selection of 64 variable wide precursor ion selection windows. The 64-variable window schema was optimized based on a normal human cell lysate sample, covering the precursor mass range of 400–1,200 m/z. The effective isolation windows can be considered as being 399.5~408.2, 407.2~415.8, 414.8~422.7, 421.7~429.7, 428.7~437.3, 436.3~444.8, 443.8~451.7, 450.7~458.7, 457.7~466.7, 465.7~473.4, 472.4~478.3, 477.3~485.4, 484.4~491.2, 490.2~497.7, 496.7~504.3, 503.3~511.2, 510.2~518.2, 517.2~525.3, 524.3~533.3, 532.3~540.3, 539.3~546.8, 545.8~554.5, 553.5~561.8, 560.8~568.3, 567.3~575.7, 574.7~582.3, 581.3~588.8, 587.8~595.8, 594.8~601.8, 600.8~608.9, 607.9~616.9, 615.9~624.8, 623.8~632.2, 631.2~640.8, 639.8~647.9, 646.9~654.8, 653.8~661.5, 660.5~670.3, 669.3~678.8, 677.8~687.8, 686.8~696.9, 695.9~706.9, 705.9~715.9, 714.9~726.2, 725.2~737.4, 736.4~746.6, 745.6~757.5, 756.5~767.9, 766.9~779.5, 778.5~792.9, 791.9~807, 806~820, 819~834.2, 833.2~849.4, 848.4~866, 865~884.4, 883.4~899.9, 898.9~919, 918~942.1, 941.1~971.6, 970.6~1006, 1005~1053, 1052~1110.6, 1109.6~1200.5 (containing 1 m/z for the window overlap). SWATH MS2 spectra were collected from 100 to 2,000 m/z. The collision energy (CE) was optimized for each window according to the calculation for a charge 2+ ion centered upon the window with a spread of 15 eV. An accumulation time (dwell time) of 50 ms was used for all fragment-ion scans in high-sensitivity mode and for each SWATH-MS cycle a survey scan in high-resolution mode was also acquired for 250 ms, resulting in a duty cycle of ~3.45 s.PrideNot availablemonohydroxylated residueProfile-mode .wiff files from shotgun data of Cal51 cells, together with those of HEK293, LNCap, U2OS and Hela cells included in the previously published SWATHatlas (34 runs in total, for the purpose of increasing the coverage of the transcript-centric spectral library used in this study)60 were all centroided and converted to mzML format using the Sciex Data Converter v.1.3 and converted to mzXML format using MSConvert v.3.04.238. The MS2 spectra were queried against the fasta file of Ensembl 66 appended with reversed sequence decoys61. Two types of search engines, xTandem62 and Omssa63, were used through iPortal interface for sophisticated proteomic workflows64. The search parameters are: static modifications of 57.02146 Da for cysteines, variable modifications of 15.99491 Da for methionine oxidations. The parent mass tolerance was set to be 30 p.p.m and mono-isotopic fragment mass tolerance was 50 p.p.m. Fully-tryptic peptides and peptides with up to two missed cleavages were allowed. The identified peptides were processed and analyzed through Trans-Proteomic Pipeline 4.5.2 (TPP)65 and were validated using the PeptideProphet score66 . All the peptides were filtered at a false discovery rate (FDR) of 1%. Spectral library generation and targeted data analysis. The raw spectral libraries were generated from all valid peptide spectrum matches for the shotgun measurement of the light peptides, and then refined into the non redundant consensus libraries59 using SpectraST67. For each peptide, the retention time was mapped into the iRT space58 with reference to a linear calibration constructed for each shotgun run, as previously described59. The MS assays constructed from Top 5 most intense transitions with Q1 range from 400 to 1200 m/z excluding the precursor SWATH window were used for targeted data analysis of SWATH maps. The whole process of SWATH targeted data analysis was carried out using OpenSWATH4. Based the spectral library generated above, OpenSWATH firstly identified the peak groups from all individual SWATH maps at a global peptide FDR=1% and then aligned them between SWATH maps (a total of 12 files including technical and biological replicates) based on the clustering behaviors of retention time in each run with a non-linear alignment algorithm68. Specifically, only those peptide peak groups identified in more than 75% samples (i.e., 9 files) were reported and considered for alignment with the max FDR quality of 0.05 (quality cutoff to still consider a feature for alignment) and/or the further constraint of less than 60 second RT difference in LC gradient after iRT normalization69. The requantification option in OpenSWATH was disabled. An initial set of 16,779 peptides was detected in all samples from multiple experiments using SWATH mass spectrometry and mapped against all the protein coding transcripts annotated in Ensembl v66, including those with a nonsense-mediated decay biotype. Removal of peptides that mapped to more than one gene led to a set of 14,695 peptides (corresponding to 2,805 genes), which was used for downstream analysis.ProteomicsRuedi AebersoldTripleTOF 5600PARTIALDepartment of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, SwitzerlandHomo Sapiens (human)liu@imsb.biol.ethz.chNot availableBiologicalInstitute of Molecular Systems Biology, ETH Zurichsodium salt, ammonium formate, 13C-labeled, IL1BC, cadmium salt, MeCN, ion, Vinegar, CG3938., fond, CASP-1, rad, Basodexan, magnesium formate, CycEI, zinc salt, Long Term, dmTAF[[II]]230, 2-Propanone, Polypeptides, protein polypeptide chains, KL receptor activity, E260, cobalt(II) formate dihydrate, CH3-C#N, B1, SCO5, Urea, DBF3|PRP8, SCO1, Il1bc, Gsfsow3, WMS, insoluble, average, Divorced, Grain, TFIID TAF250, cel, Basen, proteins, Divorces, present in organism, W, T6G21.3, Acetic Acid Glacial, IL-1BC, HPLC, AI047805, isolation and purification, allergic reaction, Dehydrated ethanol, decreased, Magics, scientific observation, D1, sample, MeCO2H, Bs, autolysin activity, z, nickel salt, SGS, spiritus vini, LC-MS2, AU019467, dTAF[[II]]230, Stars, Caspase-1 subunit p10, small interfering RNA, cobalt (+2) salt, TAF200, Longterm Effect, Normalcy, Cyc E, not genetically inherited, DmelCG7788, Absolute Alcohol, ACMICD, PBT, Glacial, sodium (4:1:1) salt, BG:DS07108.3, Dm1, magnesium salt, DmelCG42628, Base2, Base1, Raps, NICR1066, l(2)05206, [OEtH], reticulate acropigmentation of Dohi, pbt, Industrial, RP13, Industrial Arts, Ethylic acid, maintenance of localization, instrument, proportionality, formate, dyschromatosis symmetrica hereditaria 1, Dmel_CG15720, pooled, rp13, rate, cromium (+3), lysed material, beta Trypsin, Sfprp8l, alcohol etilico, Methylcarbinol, Ultrasound, Taf250, liquid chromatography-tandem mass spectroscopy, Ultrasound Imaging, Drice, bacteriolytic toxin activity, ammonium (4:1) salt, methyl cyanide, DRICE, lead (+2) salt, STARS, TAF230, krk1, nickel formate dihydrate, cycline, lead formate, Effects, DrIce, DrICE, DmcyclinE, etanol, aluminum salt, dyschromatosis symmetrica hereditaria, Carbamide, precursor, 2D-US, protein-containing complex, thomson, body system, DmelCG5692, CG7826, mass-to-charge ratio, Human, cdi7, period, cyclinE, isolation, sensitive, LC-MSMS, CG7835, Cdi7, CDI7, CG42273, system, Carmol, Medical, PH3, prpc8, Normalities, proportion, anatomical systems, dTAF[[II]]250, pins, thallium (+1) salt, Longterm, cell, ethanol, PCE-2, beta-Trypsin, rubidium salt, Long-Term, methanoic acid, DmcycE, ions, study assay, PINS, Solution, CD156, dTAF250, C2H5OH, Caspase-1 subunit p20, SNRNP220, peptidos, PTHB1, measuring, proto-oncogene c-Kit, AcOH, ultrasonography, CG5692, grupo, 1-(14)C-labeled, caspase 3, BG:DS00004.13, potassium formate, CE-2, Mischung, DSH, DSH1, copper (+2) salt, acetonitrile, buffer, ribosome-associated ubiquitin-dependent protein breakdown, Cell, dTAF230, polypeptide, l(2)k02514, native protein, DmCycE, carbonyldiamide, C18, KIT ligand receptor activity, cupric formate, chemical analysis, Long Term Effects, alcool ethylique, TAF[[II]]250/230, MFS1, aluminum formate, Pins, XKrk1, Taf[[II]]250, AU020952, hypersensitivity, lithium formate, holin, Scan Duration, l(2)k02602, underdeveloped, storage, Separations, Striated muscle activator of Rho-dependent signaling, hydrogen phosphorus, IL-1 beta-converting enzyme, ethanenitrile, Interleukin-1 beta-converting enzyme, CD117, CD156a, Longterm Effects, ME-IV, D-CycE, hydrogen phosphide, MeCOOH, ACETIC ACID, C-Kit, Ssm, CG4346, xkl-1, quotient, liquid chromatography tandem mass spectrometry, nucleobases, sequestering, Ethanoic acid, liquid chromatography tandem mass spectroscopy, Grain Alcohol, NCMe, determination, selection process, instrument configuration, zinc formate, CG7788, crice, Essigsaeure, lead salt, protein, Xkl-1, CG15720, lysis, 1-hydroxyethane, alcohol, peptide, Ethanol, Gsfsco1, ammonium tetraformate, ClvPrd, peptido, Ccne, Glacial Acetic Acid, Alkohol, Min, protein aggregate, present in fewer numbers in organism, Gsfsco5, Effect, DmelCG42273, SOW3, rsh, LCMSMS, reference sample, peptides, E927b, Ethyl, bases, E430039A18Rik, hypoplasia, Acetic Acid, min, mAPC, purification, Aethanol, copper, [CH2Me(OH)], SCAN, INSDC_feature:ncRNA, hprp8, retention, INSDC_qualifier:siRNA, lithium salt, ICE, ethoic acid, necrosis, Sl, GPHYSD2, ammonium (2:1) salt, Long-Term Effects, Dmel_CG4346, ratio, symmetric dyschromatosis of the extremities, 2-Dimensional Ultrasound Imaging, prp-8, wide/broad, Alcohol, ice, iCE, drice, Nucleobase, Arts, LC-MS/MS, Dmel_CG7826, Tr-kit, Th, Ethyl alcohol, TAFII-250, TAF250/230, br37, Thermo Scientific, ur, US, any method, carbamide, TAFII250, Ionen, drIce, drICE, kl1-A, bacteriocin activity, overlap, KIT, ETHANOL, nickel (+2) salt, Acetic acid, Dmel_CG7835, Mnb, MNB, parent ion, tyrosine-protein kinase Kit, ammonium salt, 2-Dimensional Grayscale Ultrasound Imaging, EtOH, MS/MS, ribosome-associated degradation, D11Bwg0410e, P45, familial reticulate acropigmentation of Dohi, cobaltous formate, ribosome-associated ubiquitin-dependent protein degradation, Ultrasonography, kit, MASS, AW124434, CG17603, TAF[[II]], REM3, organ system, H2NC(O)NH2, wide, Health, lysin activity, l35Dd, precursor ion, SR3-5, Acetamide, Human 细胞, Base, copper salt, hypersensitivity reaction disease, p45, Polypeptide, RAD1, 1728, time, Gruppe, base, d230, E-260, CAL-51, cesium salt, SCF receptor activity, rapsyn, FBN, ULTRASOUND, CH3-COOH, dTAFII250, cycE, broad, EfW1, l(2)br37, LC-MS-MS, CYCLE, Buffer, formic acid, Karbamid, Aethylalkohol, Methanecarboxylic acid, Human cell, potassium salt, polypeptide chain, reduced, scfr, dmTAF1, Ms1, Taf230, subnumerary, ECTOL1, 14C-labeled, Harnstoff, Ultrasound Test, tiny, SCFR, Separated, sensitivity, CH3CO2H, Pro-Mega, TAF250, Taf200, Fdc, CYCE, MS1, Prp8, Absolute, MS2, iones, DmelCG3938, CyclE, PRP8, phosphane, 3938, Taf1p, decreased number, calcium formate, OCTD, l(2)k05007, dm-cycE, lysate, grupos, male sterility 1, ribosome-associated ubiquitin-dependent protein catabolism, cromium (+3) salt, Long-Term Effect, hypersensitivity reaction, TAF, 2-iodo-, sodium formate, Controlled, prp8, small, Controlling, DYRK1, TAF[[II]]250, CG42628, nickel formate, 3H-labeled, protein complex, strontium formate, l(3)84Ab, acropigmentation of Dohi, strontium salt, Peptide, group, LC/MS/MS, HOAc, natural protein, p230, Protein, HPRP8, TFIID, Dyrk1, CES2A1, CyeE, connected anatomical system, ACETONITRILE, DBF3/PRP8, tandem MS, cyanomethane, WMS2, Ethyl Alcohol, Separation, c-KIT, hypersensitive, TAF[[II]]230, E 260, uree, MALE STERILITY 1 PROTEIN, Normality, urea, CC1, l(2)35Dd, Tripcellim, Rest, TAF[II]250, acide acetique, sample population, chromic formate, UREA, Interleukin-1 beta convertase, Trypure, hydroxyethane, c-kit, DmelCG17603, Ion, PRPC8, ultrasound, SSKS, 3.4.22.36, Calibrations, calcium salt, INS No. 260, Peptid, RAD, Rad, assay, acetic acid, variable, CG3938, groupe, TAF1293 cell, projections, IPP2A2, Bru, Materials, dmMOF, Raw, determination, Mononucleosis, Visible Light, protein, 5730420M11Rik, DmelCG9648, peptide, Polypeptides, L-Isomer Methionine, protein polypeptide chains, Methionine, Drug Tolerance, peptido, ClvPrd, hnu, dMOF, HEK 293, Human Embryonic Kidney 293, Analysis, protein aggregate, foton, WMS, Mass Spectrum Analysis, Svc, SET, F, peptides, Analyses, TAF-I, M, dMax, phosphatase 2A inhibitor I2PP2A, E430039A18Rik, chemical analysis., mononucleosis, proteins, behavioral response to stimulus, DmelCG4299, set, IGAAD, glandular fever, DmelCG10574, papilla, s, Consensus Development, Gammaherpesviral mononucleosis, GPHYSD2, Racemethionine, drug tolerance, SGS, gamma, ratio, single-organism behavior, 293 HEK, phapii, LNCaP, Radiation, anatomical protrusion, Process, immune system tolerance, 2-Amino-4-(methylthio)butyric acid, lamina, CG9648, StF-IT-1, Maps, flanges, Th, Light, U-2 OS, Search, INSDC_feature:misc_RNA, Spectrum Analysis, Acceptance Processes, ACMICD, bHLHd7, bHLHd6, Spectroscopy, bHLHd9, LNCAP, bHLHd8, Acceptance Process, bHLHd5, LIGHT, bHLHd4, shelf, dmax, Immunologic Tolerance, Genetic Materials, Methionin, methionine, Hmet, Genetic Material, Visible Radiations, parent ion, Visible Radiation, HLA-DR-associated protein II, HVEML, DI-2, 293, MS/MS, I-2Dm, shelves, proportionality, Spectrometry, rate, INSDC_feature:gene, MASS, CG4299, inhibitor of granzyme A-activated DNase, projection, ridge, infectious mononucleosis, I-2PP1, MOF, Mof, light quantum, Self Tolerance, TAF-IBETA, Tolerance, precursor ion, spine, Material, Cells, Filatov's disease, Cistron, TAF-Ibeta, Polypeptide, Liquimeth, i2pp2a, Pfeiffer's disease (disorder), U2OS, 2-amino-4-(methylsulfanyl)butanoic acid, False, lamellae, Processes, CAL-51, LNCAP cell, L Isomer, FBN, Gene, Development, precursor, Spectrum Analyses, thomson, protein-containing complex, aligned, process of organ, PHAPII, Ly113, mass-to-charge ratio, protrusion, lamella, DmelCG3025, Mono, template-activating factor I, polypeptide chain, ECTOL1, Consensus, Mass, L-Methionine, Del(8)44H, light, Pedameth, Mass Spectroscopy, Gammaherpesviral mononucleosis (disorder), study, Search Engines, proportion, MAX, Genetic, MS2, Lichtquant, HeLa, ipp2a2, 2pp2a, TPP, ridges, Visible, CG10574, CD156, OCTD, max, 2-amino-4-(methylthio)butanoic acid, 2PP2A, taf-ibeta, Infectious Mononucleosis, Pfeiffer's disease, dSET, dSet, photon, peptidos, TR2, Behaviors, metionina, laminae, behaviour, data, DL-Methionine, protein complex, anatomical process, igaad, CG3025, aligned to, backward, L-Isomer, CD258, Cistrons, Cell, Peptide, Engine, AI875693, polypeptide, HeLa Cell, Immune Tolerance, native protein, natural protein, I-2PP2A, Dm I-2, Protein, I2PP2A, sequence, human embryonic kidney cell, MFS1, Kissing disease, Library, tandem MS, flange, Mass Spectrum Analyses, Met, WMS2, organ process, Radiations, centric, Acceptance, Mass Spectrum, Col4a-1, HVEM-L, Photoradiation, HEK293, Glandular Fever, HEK-293, CD156a, primary structure of sequence macromolecule, LTg, processes, process, dSET/TAF-Ibeta, 2610030F17Rik, Photoradiations, behavioural response to stimulus, Data, SSKS, Monocytic angina, AA960152, Calibrations, alpha-amino-gamma-methylmercaptobutyric acid, quotient, U2-OS, monocytic angina, processus, Peptid, Glandular fever, assay, variable, AA407739, Data Analyses, reversed, Immunological Tolerancenuclear mRNA splicing via U2-type spliceosome, other disease, Ribonucleic, CDF, human being, Transcriptome Profile, nucleocapsid, number, Gene Expression Profile, via spliceosome, Gene, ZDBF1, protein, Profiles, Spectrum Analyses, protein-containing complex, composed of, presence, Transcripts, Alternative, method, protein polypeptide chains, POF, polypeptide chain, reduced, diseases, nuclear mRNA splicing, spliceosome, subnumerary, Messenger, DmelCG1768, method used in an experiment, Alternative RNA Splicing, Gene Products, HILDA, Mass, symptoms, disease or disorder, diseases and disorders, DBF3|PRP8, AGAMOUS-like 61, Analysis, protein aggregate, present in fewer numbers in organism, Non Polyadenylated, prpc8, RNA Gene Products, DiA, Mass Spectroscopy, pre-mRNA splicing, Mass Spectrum Analysis, Nested Transcripts, human disease, Gene Expressions, l(2)k07135, Genomes, Analyses, Prp8, Profile, PRP8, RNA Splicing, MAPKKK5, composition, proteins, Ask, ASK, DIASP, decreased number, Expressions, man, hprp8, Signatures, CHIF, DIA, Dia, retention, Non-Polyadenylated RNA, non-neoplastic, Transcript, 4-(4-dihexadecylaminostyryl)N-methylpyridium iodide, decreased, Expression Signature, 38E.16, Transcriptomes, Mekk5, SNRNP220, disorder, Homo sapiens disease, Expression, DIA2, spliceosome complex, Alternate Splicings, prp8, AU019467, screening, RNA, data, prp-8, findings, Transcriptome, splicing GT-AG intron, ribose nucleic acid, protein complex, Desmoplastic astrocytoma of infancy, Expression Profiles, ribonucleic acids, disorders, total expressed protein, RNS, splicing AT-AC intron, CG1768, compositionality, DRF2, INSDC_feature:misc_RNA, Spectrum Analysis, Splicings, Spectroscopy, RNA Splicings, Gene Expression, Splicing, count in organism, ms(2)04138, Alternative RNA Splicings, Desmoplastic infantile astrocytoma, INSDC_feature:intron, native protein, natural protein, yeast nucleic acid, Ribonukleinsaeure, Expression Signatures, HPRP8, MLPLI, Protein, Gene Expression Signatures, Diseases, core, pentosenucleic acids, condition, Ribonucleic acids, Gene Expression Signature, Dias, Expression Profile, Alternate Splicing, DBF3/PRP8, Mass Spectrum Analyses, Nested Transcript, Transcriptome Profiles, 7420452D20Rik, ribonucleic acid, Acid, Mass Spectrum, pre-mRNA splicing factor activity, AA545217, RP13, Nested, DBF4A, maintenance of localization, storage, D11Bwg0410e, content, Non Polyadenylated RNA, POF2, F27C12_24, F27C12.24, Non-Polyadenylated, Spectrometry, signs, mRNA splicing, nuclear mRNA splicing via U12-type spliceosome, rp13, Ribonucleic Acid, Sfprp8l, human, Alternate, plan specification, disease, Alternative Splicings, ASK1, Gene Expression Profiles, PRPC8, structure, DIANA, Alternative RNA, Spliceosome, medical condition., quantitative, sequestering, Signature, Proteomes, presence or absence in organismNested Transcripts, human being, Nested, RNA Splicing, total expressed protein, Ximpact, man, impact-a, human, Alternate, Transcripts, Splicings, Transcript, RNA Splicings, Alternative, Splicing, Alternative RNA Splicings, Alternative Splicings, Proteomes., Alternative RNA Splicing, Alternative RNA, E430016J11Rik, RWDD5, Alternate Splicing, Alternate Splicings, Nested Transcript325trueImpact of alternative splicing on the human proteomeAlternative splicing is a critical determinant of genome complexity and by implication, is assumed to engender proteomic diversity. This notion has not been experimentally tested in a targeted, quantitative manner. Here, we have developed an integrative approach to ask whether dynamic perturbations in mRNA splicing patterns that follow depletion of the core spliceosome factor PRPF8 alter the composition of the proteome. We integrate RNA-sequencing (to comprehensively report intron retention, differential transcript usage and gene expression) with a newly developed data-independent acquisition (DIA) method, SWATH-MS (Sequential Window Acquisition of all THeoretical spectra – mass spectrometry), to capture an unbiased and quantitative snapshot of constitutive and alternative splicing events at the protein level. Whereas intron retention is accompanied by decreased protein abundance, dynamic alterations in differential transcript usage and gene expression alter protein abundance proportionally to transcript levels. Our findings exemplify how RNA splicing exerts a pervasive effect linking isoform expression in the human transcriptome with proteomic diversity, and provide a toolkit for studying its perturbation in human diseases.2017-08-022015-12-04PXD003278280655164609918049829535851114514621333524422492156843179819354407110946194202452787284218809036809327042933084581058113633363515242601198343006636362758385696363NCBITaxon:1280688885618961114301187263514725557114526314526236493461364031952065126957366649239161974670738538836411354615821582310665903416889539053113636476272112329442229118099555832323122586365458333460792519365914320597129386558195851149315242592637371250232NCBITaxon:127211195774818661968211488584310430024513586722568076175543735925408169180454266834295319453058612668352010891498225058149636744521585545281132081479564100392375146369420494332089643795042089634459741379791387127283844006672032754532602935293671567593880364784346438677160521659940483216597216595455032309813152826930421659156271616968339162432183217546271292042779447623764543121810017203094980197525556978159509329629439843948840479NCBITaxon:21575253381315273551531051067439899037122857759515205968762262311210884555588845587077918178081736309431796758144571698936160488409859NCBITaxon:37162104782679401505907148364320421061320491381693630355543204663263331199456545674535110254111032174592161292886712642012886764100264203694569410167634102107612773515248671316582392547279334281461358430141729876091746334577444916754760489321287356756840301350621917265181301941119294393671104113167342810239320255598632582788396591047594120347495109716049016529982790513355252731336636666681024298036691758543980110243260866310245980875741755518320196447145268012675777641457966313670190832331156114155355104219325552113834396571927634981812359871069955529NCBITaxon:4751850298335476113615741465477180066556484337677602072144137929976798251021699823897641400820840741400821137351155711089725482444474153NCBITaxon:505578996984467525100036465349148624633414714749354801946751299114359741276473137081154666326547876565893639835322159589366983819201110677577271034811208370985234878026555414884142228638096647946409860995861571871766859919633715729513722117110129292117527110768783497419860202809134858513485845025392499148840633424951869123423515233598589859886559132497464998703920211107294331171139363527215987419085037117082711708282874341821144522350241451139692931902197822459390774636046147123747226940210195375939723287581721874202532566481219071NCBITaxon:573905079452659600131972211915853101774208588596321387NCBITaxon:61575052102830798951558641105198997237505998971246663576131720002631887634196102642661088878176179212190659407121906772275885815689964975151950582873756269072445066NCBITaxon:250682023717NCBITaxon:1703612201999857050605061724074767927450627249159615333554319313355418400524643213686150596487844612286662941786661347515363291018194301086731018510184NCBITaxon:176357303963558633149246437286287151458670580430801321650852718481865368595105135553482260710185687294296110091416333NCBITaxon:619184023210007348824123363630710470058127187878645159812894555663577553470951451225786425011449447260704NCBITaxon:1335626260705260707573061658457367734054163677483167233150561007133705654195NCBITaxon:3055504457913998781681190119287512987222829126806315327341478281055524NCBITaxon:9606135858469008171929111167815021979153283051930283672859300852900561742NCBITaxon:56938268830640179469913899591510118499526225435258759131515901448242838111394422584416041937047592721204414370860423281677285035158199011951953259084126738637013702492405360883858162248839432293653186156227748817562615835583715371633376057267119350199808611935021678791085791371137127240719065037147591713291212266262981288057125627457572701329090309031133355037281424337385794792491007875282411082NCBITaxon:961572433594633185862607991612432141262200298147802492670NCBITaxon:12637192812492125283038568385695911692661006581373530963223756127504542666255151978813566381647520375064422366950236009412934979054632245552536352472140125751511519976499085151514389923745461703747445446415226473243235937602647315920222887051580595536100199955508815793271602938515781149786338924794362938211620894535936479437509173NCBITaxon:6854412754225117752991590109013416738715895521012966103061296714236871585158451584915759237659393414103843271591002263767338891479235904614234442345467001287689591198332966109149446704146479293657616428034116022164133159928038280353690910891768814922999089462181214108034817939246402878892950271131332952828294456861749346819112652699722732189038210286210760124719040072715973699782811431140206411981087135547743658142450722953379698204232124223129163218033698223470871453720916457332153581575462669402656194013716546759928777020206032811431933829355083329129241292211486437457252649997283035739087183087267271037908550717629970767892683704155737292140531562887651388294128160783956392431426265489822737756115925390596207322086595882735551693605330797255181135491432137159749535026115955614321384060725878141447335215094933491949634016143347280059360463164610466076290338151845474485003567097477265669118077195124017484185560133575554792042741580536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