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 recovers 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, different conditions of reduction [dithiothreitol (DTT) and tributylphosphine] and alkylation [vinylpyridine, acrylamide and iodoacetamide] were compared. 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 electrophoresis and blotted onto polyvinylidenedifluoride (PVDF) membrane were established to achieve complete recovery of alkylated Cys. Even with the optimal on-blot iodoacetamide alkylation, there may still be some acrylamide adducts present and these were able to be separated by HPLC along with the other 16 amino acids. The Cys content has been successfully determined by Fmoc-amino acid analysis of PVDF-blotted proteins separated by 1D or 2D gel electrophoresis. Lysine alkylation with iodoacetamide and acrylamide has also been characterised. Protein identification using amino acid composition including Cys has been introduced.
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
J X
JX
Yan
W C
WC
Kett
B R
BR
Herbert
A A
AA
Gooley
N H
NH
Packer
K L
KL
Williams
D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D003545:Cysteine / Q000032:analysis*; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005449:Fluorenes / Q000032:analysis; D006868:Hydrolysis; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D010720:Phosphines; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D013439:Sulfhydryl Reagents
D000596:Amino Acids; D005449:Fluorenes; D008567:Membranes, Artificial; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010720:Phosphines; D011506:Proteins; D013439:Sulfhydryl Reagents; C043255:tri-n-butylphosphine; D003545:Cysteine; D004229:Dithiothreitol