title: Probing the reactivity of S-S bridges to acrylamide in some proteins under high pH conditions by matrix-assisted laser desorption/ ionisation.
abstract: There is compelling evidence to suggest that cysteine-acrylamide adduct formation is a modification experienced by proteins separated by two-dimensional (2-D) gel electrophoresis. Whether the -SH group involved in such complexation is offered by a free or initially disulphide-linked cysteine residue remains an open question. To address this question a number of proteins containing free and/or disulphide-linked cysteine (Cys) residues have been incubated with acrylamide monomer and examined by delayed extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). These data provide strong evidence to suggest that the presence of free Cys in the investigated proteins is not the most important requirement for the observation of Cys-acrylamide adducts. Unambiguous confirmation of this deduction was obtained by analysing the tryptic digests of the same proteins by reflectron MALDI-TOF. The assignment of the adduction sites was facilitated by the mass accuracy attained for the monitored tryptic fragments and their agreement with the corresponding predicted masses reported in the Swiss-Prot database. The same data suggest that at high pH the cysteine pairing is flexible enough to allow initially S-S linked residues to complex with acrylamide. It is also plausible that the -NH(2) terminal blockage so often encountered in proteins electroblotted from 2-D maps could originate from carbamylation, and might not have anything to do with alkylation by free, unreacted acrylamide in polyacrylamide gels.
authors.affiliation: Glaxo Wellcome Medicines Research Center, via Fleming 4, Verona, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Bordini
authors.forename: M
authors.initials: M
authors.lastname: Hamdan
authors.forename: P G
authors.initials: PG
authors.lastname: Righetti
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013455:Sulfur
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D020106:Acrylamide; D013455:Sulfur