Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is
widely practiced; ho
wever, variations providing cutting-edge RPLC performance havegenerally not been adopted even though their benefits are
well established. Here,
we describe an automated format20 kpsi RPLC system for proteomics
and metabolomicsthat includes on-line coupling of micro-solid phase extraction for sample loading
and allo
ws electrospray ionizationemitters to be readily replaced. The system uses 50
mi.d. × 40-200 cm fused-silica capillaries packed
with1.4-3-
m porous C18-bonded silica particles to obtainchromatographic peak capacities of 1000-1500 forcomplex peptide
and metabolite mixtures. This separationquality provided high-confidence identifications of >12 000different tryptic peptides from >2000 distinct
Shewanella oneidensis proteins (~40% of the proteins predictedfor the
S. oneidensis proteome) in a single 12-h ion trapt
andem mass spectrometry (MS/MS) analysis. The proteinidentification reproducibility approached 90% bet
weenreplicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min,
and 1207proteins
were identified in 120 min through assignmentof 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS
wasapproximately 10
6 based on analyses of a human bloodplasma sample, for
which 835 distinct proteins
wereidentified
with high confidence in a single 12-h run. Asingle run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomicssample.