β‐elimination: An unexpected artefact in proteome analysis

Abstract

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.

A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation

“…Although enzymatic liberation of O-linked glycans can be achieved with a combination of several glycosidases, there is no counterpart to pNGase-F for O-linked glycoproteins. Chemical removal of O-linked glycans can be achieved under alkaline conditions (21), but base-catalyzed ␤-elimination is not specific to mucin-type O-linked glycans and also cleaves other posttranslational modifications [i.e., phosphates (22,23), ␤-OGlcNAc (24), and thiols (25)]. In addition, the harsh conditions used for ␤-elimination can damage polypeptide backbones (26).…”

Purity profiling of Peptide Drugs

“…Basecatalyzed cleavage of the C-S bond in Cys results in dehydroalanine and thiocysteine, which both degrade further. Dehydroalanine is unstable and susceptible to electrophilic addition reactions, whereas thiocysteine degrades to free thiol groups [91,100,101].…”

Alkaline cleavage of covalent bonds in chicken insulin and bovine ?-lactalbumin analyzed by matrix-assisted laser desorption/ionization- mass spectrometry

“…A MALDI-TOF mass spectrum of lysozyme treated at pH 13.0, 257C, for 6 h in the presence of 0.1% DTT. The mass (m/z) values were calibrated using the average masses of (reduced lysozyme 1 H) 11 and (reduced lysozyme 1 2H) 21 . Figure 2 shows the zoomed regions around the major peaks in Fig.…”

“…In the course of re-examining the extraction method for direct mass spectrometric analysis of the proteins on CBB-stained PAGE gels, we noticed that MS should be useful to analyze the effects of alkaline hydrolysis or b-elimination reaction on proteins during the alkaline treatment. Further, Herbert et al [11] recently found, using MALDI-TOF-MS, the fragmentation of lysozyme during IEF in solution and claimed that it is caused by b-elimination reactions of the protein at the alkaline pI position.…”