Tandem mass spectrometry methods for definitive protein identification in proteomics research.
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.
Keough
T
T
The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, OH, USA. keough.tw@pg.com
Lacey
M P
MP
Fieno
A M
AM
Grant
R A
RA
Sun
Y
Y
Bauer
M D
MD
Begley
K B
KB
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D020543:Proteome