Processing complex mixtures of intact proteins for direct analysis by mass spectrometry.
For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
Fanyu
F
Meng
Benjamin J
BJ
Cargile
Steven M
SM
Patrie
Jeffrey R
JR
Johnson
Shaun M
SM
McLoughlin
Neil L
NL
Kelleher
D000465:Algorithms; D000595:Amino Acid Sequence; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013501:Surface-Active Agents
D011506:Proteins; D013501:Surface-Active Agents