Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry.
Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10mM and/or reaction time to below 5min. Maximal removal of Cys activity was observed in tissue homogenates at 40mM NEM within 1min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.
School of Molecular Bioscience, The University of Sydney, 2006, Australia.
Jana
J
Paulech
Nestor
N
Solis
Stuart J
SJ
Cordwell
D000478:Alkylation; D000818:Animals; D001923:Brain Chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011917:Rats, Inbred Lew
D009419:Nerve Tissue Proteins; D010455:Peptides; D003545:Cysteine