Characterization of low abundant membrane proteins using the protein sequence tag technology.
About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature. In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions. The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins. The identification of these N-terminal PST peptides is based on LC-MS/MS. The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment. The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins. More interestingly, among these membrane proteins 56% are predicted to be of low abundance. These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.
Proteome Sciences R&D, Industriepark Höchst, Building G865a, D-65929 Frankfurt am Main, Germany.
Thorsten
T
Prinz
Jörg
J
Müller
Karsten
K
Kuhn
Jürgen
J
Schäfer
Andrew
A
Thompson
Josef
J
Schwarz
Christian
C
Hamon
D002254:Carbonates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003062:Codon / Q000235:genetics; D003488:Cyanogen Bromide / Q000737:chemistry; D030562:Databases, Protein; D015870:Gene Expression / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics
D002254:Carbonates; D003062:Codon; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; C005686:sodium carbonate; D003488:Cyanogen Bromide