Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
26975722
DOI
10.1016/j.jprot.2016.03.016
PII: S1874-3919(16)30069-0
Knihovny.cz E-resources
- Keywords
- CNBr, Hydrophobicity, Integral membrane proteins, Lymphoma, Missing proteins, Transmembrane,
- MeSH
- Chromatography, Liquid methods MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Humans MeSH
- Lymphoma, Mantle-Cell chemistry MeSH
- Membrane Proteins analysis MeSH
- Neoplasm Proteins analysis MeSH
- Peptides analysis MeSH
- Proteome chemistry MeSH
- Proteomics methods MeSH
- Tandem Mass Spectrometry methods MeSH
- Trypsin chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Membrane Proteins MeSH
- Neoplasm Proteins MeSH
- Peptides MeSH
- Proteome MeSH
- Trypsin MeSH
UNLABELLED: Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease "shaving" of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original "hppK" method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1-16 transmembrane segments. This method allowed us to identify 13 "missing proteins" - proteins with no previous evidence on protein level. BIOLOGICAL SIGNIFICANCE: Integral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called "missing proteins".
BIOCEV 1st Faculty of Medicine Charles University Prague Czech Republic
Protemics Core Facility Faculty of Science Charles University Prague Czech Republic
The Sainsbury Laboratory Norwich Research Park Norwich UK
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