Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine beta-lactoglobulin B.
Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.
Curcuruto
O
O
Glaxo Wellcome Medicines Research Center, Verona, Italy.
Bordini
E
E
Rovatti
L
L
Hamdan
M
M
D000178:Acrylamides; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D006868:Hydrolysis; D007782:Lactoglobulins / Q000032:analysis*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D000178:Acrylamides; D007782:Lactoglobulins; D014357:Trypsin