Showing papers by "Jimmy K. Eng published in 1998"

High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry.

Abstract: We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary. The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.

Method To Compare Collision-Induced Dissociation Spectra of Peptides: Potential for Library Searching and Subtractive Analysis

Abstract: We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra The query spectrum is compared by using both mass information and fragmentation patterns Fragmentation patterns are compared to each other using a correlation function To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types Accurate and reliable comparisons are demonstrated in both types of analyses The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than compa