Tandem mass spectrometry methods for definitive protein identification in proteomics research

Citation
T. Keough et al., Tandem mass spectrometry methods for definitive protein identification in proteomics research, ELECTROPHOR, 21(11), 2000, pp. 2252-2265
Citations number
38
Categorie Soggetti
Chemistry & Analysis
Journal title
ELECTROPHORESIS
ISSN journal
01730835 → ACNP
Volume
21
Issue
11
Year of publication
2000
Pages
2252 - 2265
Database
ISI
SICI code
0173-0835(200006)21:11<2252:TMSMFD>2.0.ZU;2-J
Abstract
Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been a pplied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized pe ptides were sequenced using matrix-assisted laser desorption/ionization (MA LDI) post-source decay (PSD) and electrospray ionization-tandem mass spectr ometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spec tra of the derivatized peptides usually allow much more specific protein se quence database searches than those obtained without derivatization. We als o report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass s pectra provide predictable fragmentation patterns for arginine-terminated p eptides. The spectra are easily interpreted de novo, and they facilitate er ror-tolerant identification of proteins whose sequences have been entered i nto databases.