Fast on-column protein digestion with subsequent peptide mapping using tandem mass spectrometry with information dependent acquisition.
A platform for rapid on-line protein digestion of protein mixtures for direct infusion to a mass spectrometer is presented. A mixture of protein A, staphylococcal enterotoxin B and cytochrome c was used as a model mixture injected on a gel filtration column and a trypsin reactor which were connected in series to a micro liquid chromatography (microLC) system. The peptides in the column eluate were analyzed with ESI tandem mass spectrometry, utilizing information dependent acquisition (IDA). In one step, the proteins in the mixture (microM concentrations) were concomitantly desalted, separated, digested and identified with an overall analysis time of less than 40 min. Protein sequence coverage of 78-95% for the involved substances was achieved.
Hedström
Martin
M
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Andersson
Maria
M
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009862:Online Systems; D010449:Peptide Mapping / Q000379:methods*; D013205:Staphylococcal Protein A / Q000302:isolation & purification*
D013205:Staphylococcal Protein A