Global proteome discovery using an online three-dimensional LC-MS/MS.
We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.
Wei
Jing
J
Diversa Corporation, 4955 Directors Place, San Diego, California 92121, USA. jwei@diversa.com
Sun
Jun
J
Yu
Wen
W
Jones
Arianna
A
Oeller
Paul
P
Keller
Martin
M
Woodnutt
Gary
G
Short
Jay M
JM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D009862:Online Systems; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate; D014508:Urea
D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D012967:Sodium Dodecyl Sulfate; D014508:Urea