Beta-elimination: an unexpected artefact in proteome analysis.
Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.
Herbert
Ben
B
Proteome Systems, North Ryde, Sydney, NSW, Australia. ben.herbert@proteosystems.com
Hopwood
Femia
F
Oxley
David
D
McCarthy
John
J
Laver
Matt
M
Grinyer
J
J
Goodall
A
A
Williams
Keith
K
Castagna
Annalisa
A
Righetti
Pier Giorgio
PG
D000478:Alkylation; D016477:Artifacts; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009113:Muramidase / Q000032:analysis*; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D018892:Proton-Motive Force; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D010455:Peptides; D020543:Proteome; D009113:Muramidase; D003545:Cysteine