Summary
Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.
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Abbreviations
- dptu:
-
diphenylthiourea
- dmptu:
-
dimethylphenylthiourea
- prop-cys:
-
S-propylcysteine
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Hale, J.E., Butler, J.P. & Pourmand, R.R. Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents. Amino Acids 10, 243–252 (1996). https://doi.org/10.1007/BF00807326
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DOI: https://doi.org/10.1007/BF00807326