On-line counting of cysteine residues in peptides during electrospray ionization by electrogenerated tags and their application to protein identification.
The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human alpha-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.
Dayon
Loïc
L
Laboratoire d'Electrochimie Physique et Analytique, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédéralede Lausanne, Lausanne, Switzerland.
Roussel
Christophe
C
Prudent
Michel
M
Lion
Niels
N
Girault
Hubert H
HH
D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000032:analysis*; D006801:Humans; D007768:Lactalbumin / Q000032:analysis / Q000302:isolation & purification; D009862:Online Systems; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D010455:Peptides; D011506:Proteins; D012710:Serum Albumin, Bovine; D007768:Lactalbumin; D003545:Cysteine