GPMDB00306OtherXiaofeng Guo, et al.InstrumentNot AvailableNot availableHomo_sapiens_viruses, Human_femaledavid.trudgian@utsouthwestern.edu24696503UT Southwestern Medical Centerhttp://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026988http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026987http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026998http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026986http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026994http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026993http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026992Not availableGPMDBBottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases.Confetti: a multiprotease map of the HeLa proteome for comprehensive proteomics.Guo Xiaofeng X,Trudgian David C DC,Lemoff Andrew A,Yadavalli Sivaramakrishna S,Mirzaei Hamid H,Guo Xiaofeng X, Trudgian David C DC, Lemoff Andrew A, Yadavalli Sivaramakrishna S, Mirzaei Hamid HHeLa, RUTBC3, rabGAPLP, RabGAP-5, RCB0007, MUTYH-Associated Polyposis, MYH-Associated Polyposis, RUSC3, MAP, Proteomes., RABGAP5IGF-I, APR, data, steel factor, mol, LRP, Slf, Biocatalysts, hematopoietic growth factor KL, Proteins, Somatomedin-C, somatomedin, APOER, Gene, FPH2, enzyme activity, LysY, electronic data file, collisionally activated dissociation, Cell, sKITLG, CID, polypeptide, Polypeptides, CD91, CAD, mechano growth factor, Protein, Gene Products, computer data file, TGFBR5, AGPR, Igf-1, SHEP7, mast cell growth factor, enzymes, cell, NOXA, Stem cell factor, HeLa, ArgC, somatomedin-C, SF, Kitl, proteins, N-acetyl-alpha-glutamyl-phosphate reductase activity, lysed material., STAT5, Mast cell growth factor, Protein Gene Products, Gene Proteins, stem cell factor, NAGSA dehydrogenase activity, KL-1, lysate, A2MR, N-acetyl-aminoadipate semialdehyde dehydrogenase activity, IGF1, MGF, c-Kit ligand, Mechano growth factor, LRP1A, KITLG, RCB0007, Soluble KIT ligand, SCF, Sl, Proteomes, IGFBP3RHeLa, RUTBC3, rabGAPLP, RabGAP-5, RCB0007, MUTYH-Associated Polyposis, MYH-Associated Polyposis, RUSC3, MAP, Proteomes., RABGAP5Gene., data, posttranslational modification, use, Materials, determination, SRML1, Biocatalysts, Trypsin/K, PAPT, Proteins, number, Gene, enzyme activity, SPDSY, Cistrons, presence, Multiple Reaction Monitoring, polypeptide, peptide, Polypeptides, count in organism, count, resilient, tough, Protein, chemical analysis, Gene Products, sequence, Genetic Materials, Genetic Material, strong, posttranslational amino acid modification, PTM, Genetic, MRM, enzymes, Identification, SRM, HeLa, beta-Trypsin, Tripcellim, Identifications (Psychology), proteins, posttranslational protein modification, lysed material, beta Trypsin, primary structure of sequence macromolecule, post-translational amino acid modification, Protein Gene Products, Gene Proteins, Trypure, lysate, Material, post-translational modification, Cistron, RCB0007, assay, quantitative, Proteomes, SPS1, presence or absence in organism3006http://gpmdb.thegpm.org/~/dblist_gpmnum/gpmnum=GPM11210026998dbgap_ncbi~0patentfamilies~0rfam~0merops~0complex-portal~0uniprot~0wormbaseparasite~0embl-covid19~0reactome~0emdb~0wgs_masters~0ebiweb_resources~0opentargets_genetics~0biomodels_all~0ipd-mhc~0ebiweb_teams~0taxonomy~0genome_assembly~0sc-experiments~0ebiweb_people~0enzymeportal_enzymes~0ipd-nhkir~0cellosaurus~0pdbe~0chebi~0patentproteins~0interpro7~0uniref~0chembl~0pdbekb~0gpcrdb~0hgnc~0sc-genes~0intact~0rhea~0ebiweb_training~0alphafold~0imgt-hla~0patentnucleotides~0ensemblroot~0eva_studies~0non-coding~0europepmc~0pubmed~1identifiers_registry~0pdbechem~0hpa-covid19~0eva-variants-covid19~0biosamples~0gwas_catalog~0biotools~0tls_masters~0mesh~0coding~0sra~0opentargets~0efo~0embl-pathogen~0project~0pride~1human_diseases~0geo_datasets~0embl~0treefam~0uniparc~0ols~0dgva~0intenz~0go~0tsa_masters~0biosamples-covid19~0ebiweb_corporate~0omim~0lrg~0earlycause-molecular-sequences~0ipd-kir~0empiar~0rnacentral~0orcid_data_claims~0gpmdb~3lineage-covid19~0metagenomics~0pfam~0pride archive~1varsite~00Resource Reanalysis0.00.00.0092535471930906850.01.0falseConfetti: A Multi-protease Map of the HeLa Proteome for Comprehensive ProteomicsData from ProteomeXchange, PXD ID: PXD000900. Data file: HeLa_ArgC-Elastase_CID_2.mgf. Published as part of Mol Cell Proteomics. 2014 Apr 2 . From the Abstract: {{i}} ... In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multi-protease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly, with 42% mean sequence coverage ... {{/i}}2014-04-30GPM1121002699824696503PXD000900PXD000900