Very high pressure gradient LC/MS/MS.
A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.
Tolley
L
L
Department of Chemistry, University of North Carolina at Chapel Hill, 27599-3290, USA.
Jorgenson
J W
JW
Moseley
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D010047:Ovalbumin; D014357:Trypsin