On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.
Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.
Garzotti
M
M
Glaxo Wellcome Medicines Research Centre, Verona, Italy.
Rovatti
L
L
Hamdan
M
M
D000178:Acrylamides / Q000737:chemistry*; D001030:Apamin / Q000737:chemistry / Q000302:isolation & purification; D002116:Calcitonin / Q000032:analysis / Q000302:isolation & purification; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D016211:Transforming Growth Factor alpha / Q000032:analysis
D000178:Acrylamides; D011506:Proteins; D016211:Transforming Growth Factor alpha; D001030:Apamin; D002116:Calcitonin; D003545:Cysteine