Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed b y modifying a commercially available pump in order to achieve pressures in excess of 1200 bar (17 500 psi), A computer-controlled low-pressure mixer w as used to generate solvent gradients. Protein digests were rapidly analyze d by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible det ector. 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 C ls-modified 1.5-mum nonporous silica particles. A digest of bovine serum al bumin (BSA) was analyzed by the VHPLC system connected to a mass spectromet er in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise rati os of tryptic peaks greater than 10:1 in the base peak plot mass chromatogr am, This system was also used to analyze a proteolytic digest of a rat live r protein excised from a 2-D gel separation of a liver tissue lysate, For t his analysis, the mass spectrometer was set up to perform data-dependent sc anning (automated switching from MS mode to MS/MS mode when a peak was dete cted) for peptide sequencing and protein identification by database searchi ng. The results of this analysis are compared to an analysis performed on t he same sample using the nanoelectrospray-MS/MS technique. Though both tech niques were able to identify the unknown protein, the VHPLC method gave twi ce as many sequenced peptides as nanoelectrospray and improved the signal-t o-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.