MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v01/MSV000079025/primaryOK2000000ProteomicsEA KomivesLTQ Orbitrap Velos ETDSchizosaccharomyces Pombe (ncbitaxon:4896)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=af796439cc0f4476b375e091e705b503MassIVE20UNIMOD:108 - "N-ethylmaleimide on cysteines."UNIMOD:1 - "Acetylation."UNIMOD:28 - "Pyro-glu from Q."MOD:00719 - "A protein modification that oxygenates an L-methionine residue to one of the diastereomeric L-methionine sulfoxide residues."Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin's may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type α-lytic protease (WaLP) and an active site mutant of WaLP, M190A α-lytic protease (MaLP). We assess several relevant factors, including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. When data from separate digestions with trypsin, LysC, WaLP, and MaLP were combined, proteome coverage was increased by 101% relative to that achieved with trypsin digestion alone. To demonstrate how the gained sequence coverage can yield additional post-translational modification information, we show the identification of a number of novel phosphorylation sites in the Schizosaccharomyces pombe proteome and include an illustrative example from the protein MPD2 wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.Expanding proteome coverage with orthogonal-specificity α-lytic proteases.Meyer Jesse G JG, Kim Sangtae S, Maltby David A DA, Ghassemian Majid M, Bandeira Nuno N, Komives Elizabeth A EAl(4)17, Ce, l(4)13, Gli, Ci, Cid[Mel], Ci[D], cenH3, Peptide, CENP-A, collisionally activated dissociation, Cid, CENP-C, CID, ciD, CenpA, Polypeptides, l(4)102ABc, CAD, peptido, ci[D], Ci155, ci-D, Siah, cenpA, CenH3[Cid], CG13329, CenH3[CID], BcDNA:RE21270, DmelCG2125, CenH3, ci155, peptides, CenH3/CID, Ci/Gli, Ci/GLI, CENP-A/Cid, CENP-A/CID, CENPA, DmelCG13329, X-linked combined immunodeficiency, CI, Cenp-A, CG2125, Peptid, peptidos, Polypeptide, ETD., CenpA/CID, Ci-155, CID/CENP-A, CiDtotal expressed protein, Proteomes.l(3)Ca, Peptidomics, mCRY, titin, 26S protease, l(3)62Ca, d-titin, 0020/01, threonine endopeptidase activity, Mbh1, SLS, Sls, results, ingensin, kettin, large multicatalytic protease, alpha-lytic protease, CFU-MC, HEL-S-66, DmelCG1915, Titin, prosome, Crry, multicatalytic endopeptidase complex, anon-CREST, Ket, CFU-Mast, tricorn proteinase, D-titin, l(3)j1D7, AFCP, colony forming unit mast cell, Papers., D-Titin, CG18857, MIC10, ket, sam, sal, TLX, l(3)dre8, lens neutral proteinase, CG1915, CT41299, multicatalytic proteinase (complex), KZ, polyhedral organelle, l(3)S002001, l(3)rL182, CG18242, tricorn protease, AHUS2, TRA2.10, Mcp, MCP, alkaline protease, multicatalytic proteinase, CG18245, proteasome endopeptidase complexPost Translational Amino Acid Modification, posttranslational modification, Post-Translational Protein Modifications, Peptidomics, P62, Fission, Aminosaeure, Aminocarbonsaeure, number, cleavage, distal myopathy 2, Amino acid, A4, Gene, protein, alpha-amino acid, sci, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, protein-containing complex, supernumerary, Protein Processing, TYPE, phosphorylation, Posttranslational, DAGA4, peptide, Polypeptides, Membrane Tissues, protein polypeptide chains, Fission Yeast, peptido, polypeptide chain, integral component of membrane, Post Translational Modification, Gene Products, HOW, How, Post-Translational Modifications, MAM, protein aggregate, SCG3, fission yeast, l(3)j5D5, Post Translational, 24B, Modification, Post-Translational Protein, increased, Schizosaccharomyces pombeP, posttranslational amino acid modification, amino acids, l(3)s2612, peptides, Post-Translational, Tissues, membrane region, beta-Trypsin, Tissue, stru, stubby, proteins, l(3)S053606, posttranslational protein modification, Posttranslational Protein Processing, CG10293, Protein Modifications, Post-Translational Amino Acid Modification, l(3)j5B5, Posttranslational Amino Acid Modification, DmelCG10293, ALS21, region of membrane, Posttranslational Protein, site, peptidos, MPD2, Membrane Tissue, Post-Translational Modification, membrane, data, 0904/17, distal myopathy with vocal cord weakness, clone 2.39, protein complex, Aminokarbonsaeure, Modifications, Proteins, Phosphorylations, total expressed protein, Post-Translational Protein Modification, integral to membrane, alpha-amino acids, qkr, membranous organ component, S pombe, l(3)S090417, Peptide, alpha-amino carboxylic acids, LGMD2C, polypeptide, shortened, Post Translational Modifications, native protein, natural protein, SZ1, KH93F, Protein, whole membrane, sequence, vocal cord and pharyngeal distal myopathy, phosphorylation enrichment, Protein Modification, region, beta Trypsin., who, Amino Acid, Acid, Yeast, transmembrane, membrane of organ, PRSS, PTM, Amino acids, DMDA1, increased number, Post Translational Protein Processing, Processing, Tripcellim, Who/How, VCPDM, Amino, Membrane, beta Trypsin, primary structure of sequence macromolecule, post-translational amino acid modification, Protein Gene Products, Trypure, present in greater numbers in organism, Gene Proteins, Post Translational Protein Modification, Fission Yeasts, DMDA, Amino Acid Modification, post-translational modification, SCARMD2, cardinality, Post-Translational Protein Processing, MATR3-related distal myopathy, qkr[93F], Peptid, anon-EST:Liang-2.39, Acids, Posttranslational Modifications, Polypeptide, short, Proteomes, Posttranslational Modification, accessory0PXD001771trueddDT results from 2014 MCP alpha-lytic protease proteomics paperddDT runs of peptides from four separate protease digestions. CID for z=2, ETD for z>=3Fri Jan 30 12:35:00 GMT 2015MSV00007902524425750