Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry for monitoring alkylation of beta-lactoglobulin B exposed to a series of N-substituted acrylamide monomers.
Delayed-extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, in both linear and reflectron modes, has been used to examine the alkylation of bovine beta-lactoglobulin-bound cysteines exposed to various molar concentrations (0.5-30 mM) of acrylamide and a number of its N-substituted monomers. These measurements were conducted at pH approximately 9.5, and showed that at 0.5 mM all monomers (except N-acryloylaminopropanol) resulted in a measurable alkylation of at least one cysteine out of five. At higher concentrations (15 mM) all investigated monomers resulted in substantial alkylation which, for some, involved all five cysteine residues. Reflectron MALDI-TOF measurements of a number of alkylated protein digests revealed that, at low molar ratios, the alkylation site is influenced by the identity of the monomer. For example acrylamide and N, N-dimethylacrylamide attacked Cys(160) as well as one of the three cysteines within the tryptic fragment [102-124], while other investigated monomers did not involve Cys(160). The implications of the present data for two-dimensional (2D) gel electrophoresis, and their eventual correlation to the toxicity of the investigated monomers, are discussed.
Bordini
E
E
Glaxo Wellcome Medicines Research Center, via Fleming 4, 37135 Verona, Italy.
Hamdan
M
M
Righetti
P G
PG
D000178:Acrylamides / Q000737:chemistry; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D007782:Lactoglobulins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D000178:Acrylamides; D007782:Lactoglobulins; D010446:Peptide Fragments; D014357:Trypsin