title: Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.
abstract: 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.
  
  
  
  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".
authors.affiliation: BIOCEV, First Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: O
authors.initials: O
authors.lastname: Vit
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic.
authors.forename: P
authors.initials: P
authors.lastname: Man
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic.
authors.forename: A
authors.initials: A
authors.lastname: Kadek
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Hausner
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.
authors.forename: J
authors.initials: J
authors.lastname: Sklenar
authors.affiliation: Protemics Core Facility, Faculty of Science, Charles University in Prague, Czech Republic.
authors.forename: K
authors.initials: K
authors.lastname: Harant
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic.
authors.forename: P
authors.initials: P
authors.lastname: Novak
authors.affiliation: Thermo Fisher Scientific, Bremen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Scigelova
authors.affiliation: Thermo Fisher Scientific, Hemel Hempstead, UK.
authors.forename: G
authors.initials: G
authors.lastname: Woffendin
authors.affiliation: BIOCEV, First Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Petrak
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020522:Lymphoma, Mantle-Cell / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010455:Peptides; D020543:Proteome; D014357:Trypsin
keywords: CNBr; Hydrophobicity; Integral membrane proteins; Lymphoma; Missing proteins; Transmembrane