PNNL

Abstract

Proteomics analysis based-on liquid chromatography (LC), particularly reversed-phase LC (RPLC), is widely practiced; however, cutting-edge LC performance variations have generally not been adopted even though their benefits are well established. The two major reasons behind this general underutilization are: 1) uncertainties surrounding the extent of improvement (e.g., proteome coverage), and 2) the lack of availability of automated, robust, and convenient LC instrumentation. Here, we describe an automated format 20K psi gradient nanoscale LC system that was developed to provide improved separations and sensitivity for proteomics (and metabolomics) applications. The system includes on-line coupling of micro solid phase extraction for sample loading and allows emitters for electrospray ionization to be readily replaced. The system uses 40 to 200 cm ? 50 µm i.d. fused silica capillaries packed with 1.4- to 3-µm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1,000-1,500 for complex peptide and metabolite mixtures. This separation quality allowed high confidence identification of >12,000 different peptides from >2,000 distinct Shewanella oneidensis proteins (~ 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The reproducibility was >87% for proteins identified between replicates. The protein MS/MS identification rate average exceeded 10 proteins per minute, e.g., 1,207 proteins were identified in 120 min through assignment of 5,944 different peptides. For a human blood plasma sample that was not depleted of the most abundant proteins, 835 distinct proteins were identified with high confidence in a single 12-h run. A single run with accurate mass MS detected >5,000 different compounds from a metabolomics sample.