Jx. Yan et al., IDENTIFICATION AND QUANTITATION OF CYSTEINE IN PROTEINS SEPARATED BY GEL-ELECTROPHORESIS, Journal of chromatography, 813(1), 1998, pp. 187-200
Citations number
27
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
A simple technique is introduced to identify and quantitate cysteine (
Cys) after acid hydrolysis of protein. The technique involves using 9-
fluorenylmethyl chloroformate (Fmoc)-based amino acid analysis that re
covers all of the amino acids (asparagine and glutamine are recovered
in their acidic forms) except tryptophan. Cys adducts with acrylamide
and iodoacetamide have been observed in hydrolysates of gel-separated
proteins. To enable quantitation of Cys by amino acid analysis, differ
ent conditions of reduction [dithiothreitol (DTT) and tributylphosphin
e] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were c
ompared. Optimal conditions for on-blot reduction (125 mM of DTT, pH 8
.5, at 80 degrees C) and alkylation (0.25 M iodoacetamide, pH 8.5, at
37 degrees C) of proteins which have been separated by gel electrophor
esis and blotted onto polyvinylidenedifluoride (PVDF) membrane were es
tablished to achieve complete recovery of alkylated Cps. Even with the
optimal on-blot iodoacetamide alkylation, there may still be some acr
ylamide adducts present and these were able to be separated by HPLC al
ong with the other 16 amino acids. The Cys content has been successful
ly determined by Fmoc-amino acid analysis of PVDF-blotted proteins sep
arated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoace
tamide and acrylamide has also been characterised. Protein identificat
ion using amino acid composition including Cys has been introduced. (C
) 1998 Elsevier Science B.V. All rights reserved.