Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.
Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.
Sechi
S
S
The Rockefeller University, New York, New York 10021, USA. SechiS@erols.com
Chait
B T
BT
D020106:Acrylamide; D000477:Alkylating Agents; D000478:Alkylation; D003545:Cysteine / Q000737:chemistry*; D007460:Iodoacetamide; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis*; D011725:Pyridines
D000477:Alkylating Agents; D011506:Proteins; D011725:Pyridines; D020106:Acrylamide; C029351:4-vinylpyridine; D003545:Cysteine; D007460:Iodoacetamide