Improved identification of hordeins by cysteine alkylation with 2-bromoethylamine, SDS-PAGE and subsequent in-gel tryptic digestion.
Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of hordeins with low numbers of lysine and arginine in combination with high content of proline and glutamine complicates their identification in a common proteomic workflow, because tryptic digestion produces just a few peptides amenable for successful mass spectrometric analysis. To increase the number of cleavage sites, in this work, cysteines in hordeins were chemically modified with 2-bromoethylamine (BEA) for their conversion into aminoethylcysteines. These mimic lysine residues and are recognized by trypsin as potential cleavage sites (if not followed by a proline residue) on the C-terminal side of the modified cysteine. Small extent of side reactions (towards histidine, N-terminus of the peptide, methionine, and also here the newly discovered reaction towards aminoethylcysteine) during modification with BEA could be observed after a longer period of reaction but this did not hinder the analysis when optimal conditions were used. Application of trypsin for in-gel digestion of hordeins, previously modified chemically with BEA, provided a higher number of short peptides and their subsequent mass spectrometric analysis resulted in an improved identification of hordeins. This approach can also be used for the analysis of other similar protein groups (e.g. gliadins in wheat) or other cysteines containing proteins having a low number of lysine and arginine residues in their primary structure.
Institute of Analytical Chemistry of the Academy of Sciences of the Czech Republic, v.v.i., Veverí 97, 602 00 Brno, Czech Republic.
Helena
H
Rehulková
Martina
M
Marchetti-Deschmann
Ernst
E
Pittenauer
Günter
G
Allmaier
Pavel
P
Rehulka
D000478:Alkylation; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005021:Ethylamines / Q000737:chemistry*; D005983:Glutens / Q000737:chemistry* / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry; D010446:Peptide Fragments; D014357:Trypsin / Q000378:metabolism*
D005021:Ethylamines; D010446:Peptide Fragments; C004504:2-bromoethylamine; D005983:Glutens; D014357:Trypsin; D003545:Cysteine