Chemical cleavage-assisted tryptic digestion for membrane proteome analysis.
We have developed a simple and unbiased method for membrane proteome analysis using cyanocysteine-mediated cleavage in combination with trypsin digestion. In our previous study, application of the trypsin-based phase-transfer surfactants (PTS) protocol for membrane proteome analysis provided a substantial improvement in the identification of the membrane proteome, but the task remains challenging, because trypsin often generates peptides larger than the observable m/z range. Here, we predict computationally that the combination of Cys cleavage with tryptic digestion would be more effective than trypsin digestion alone for membrane proteome analysis. To validate this prediction, we applied a combined Cys cleavage-trypsin approach to 14 microg of Escherichia coli membrane-enriched pellet. By using two-dimensional LC-MS/MS, we identified a total of 1530 proteins, of which 667 were membrane proteins. This represents a 10% increase over the number identified with the PTS protocol using optimized trypsin-based digestion in our previous study [ Masuda , T. , et al. J. Proteome Res. 2008 , 7 ( 2 ), 731 - 40 ]. The coverage of the E. coli membrane proteome was approximately 40%, ranging from 37% to 42% in various subcategories. Further, the distribution of the number of transmembrane domains per protein was unbiased compared with that in the GenoBase database. These results indicate that the combination Cys chemical cleavage-assisted trypsin digestion protocol will be a powerful tool for membrane proteome analysis.
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
Mio
M
Iwasaki
Takeshi
T
Masuda
Masaru
M
Tomita
Yasushi
Y
Ishihama
D002458:Cell Fractionation; D002853:Chromatography, Liquid; D003198:Computer Simulation; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012107:Research Design; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D020543:Proteome; D014357:Trypsin; D003545:Cysteine