On-line capillary liquid chromatography tandem mass spectrometry on an iontrap/reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line w ith an ion trap storage/reflectron time-of-flight mass spectrometer to perf orm tandem mass spectrometry for tryptic peptides. Selection and fragmentat ion of the precursor ions were performed in a three-dimensional ion trap, a nd the resulting fragment ions were pulsed out of the trap into a reflectro n time-of-flight mass spectrometer for mass analysis. The stored waveform i nverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collisi on-induced dissociation. Tandem mass spectra of various doubly charged tryp tic peptides were investigated where a conspicuous y ion series over a cert ain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by da tabase search using the sequence tag approach. Several peptides from trypti c digests of horse heart myoglobin and bovine cytochrome c were selected fo r tandem mass spectrometry (MS/MS) where it was demonstrated that the prote ins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimens ional gel separation of a human esophageal adenocarcinoma cell line. (C) 20 00 American Society for Mass Spectrometry.