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Tandem mass spectrometry methods for definitive protein identification in proteomics research
Author(s) -
Keough Thomas,
Lacey Martin P.,
Fieno Angela M.,
Grant Raymond A.,
Sun Yiping,
Bauer Mark D.,
Begley Karen B.
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(20000601)21:11<2252::aid-elps2252>3.0.co;2-o
Subject(s) - chemistry , protein mass spectrometry , chromatography , bottom up proteomics , top down proteomics , mass spectrometry , isobaric labeling , tandem mass spectrometry , electrospray ionization , tandem mass tag , sample preparation in mass spectrometry , proteome , proteomics , quantitative proteomics , biochemistry , gene
Optimized procedures have been developed for the addition of sulfonic acid groups to the N ‐termini of low‐level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two‐dimensional (2‐D) gel electrophoresis. The derivatized peptides were sequenced using matrix‐assisted laser desorption/ionization (MALDI) post‐source decay (PSD) and electrospray ionization‐tandem mass spectrometry 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 spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization‐tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine‐terminated peptides. The spectra are easily interpreted de novo , and they facilitate error‐tolerant identification of proteins whose sequences have been entered into databases.