IDENTIFICATION AND QUANTITATION OF CYSTEINE IN PROTEINS SEPARATED BY GEL-ELECTROPHORESIS

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.