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Title: Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1

Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to “mine” information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976more » were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.« less
Authors:
 [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [4] ;  [5]
  1. Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
  2. Univ. of California, Los Angeles, CA (United States). Microbiology, Immunology, and Molecular Genetics
  3. Univ. of California, Los Angeles, CA (United States). Dept of Chemistry and Biochemistry, Univ. of California, Los Angeles, CA (United States). Dept. of Biological Chemistry and UCLA-DOE Inst. for Genomics and Proteomics
  4. Univ. of California, Los Angeles, CA (United States). Microbiology, Immunology, and Molecular Genetics and UCLA-DOE Inst. for Genomics and Proteomics
  5. Univ. of California, Los Angeles, CA (United States). Dept. of Biological Chemistry and UCLA-DOE Inst. for Genomics and Proteomics
Publication Date:
OSTI Identifier:
1192899
Grant/Contract Number:
FG02-08ER64689
Type:
Accepted Manuscript
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 6; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Research Org:
Univ. of California, Los Angeles, Los Angeles, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES S-layers; archaeal surface proteins; Methanosarcina mazei; prokaryotic glycosylation; membrane proteins; concanavalin A