Enhanced electron transfer dissociation through fixed charge derivatization of cysteines.
Electron transfer dissociation (ETD) has proven to be a promising new ion activation method for proteomics applications due to its ability to generate c- and z-type fragment ions in comparison to the y- and b-type ions produced upon the more conventional collisional activation of peptides. However, low precursor charge states hinder the success of electron-based activation methods due to competition from nondissociative charge reduction and incomplete sequence coverage. In the present report, the reduction and alkylation of disulfide bonds prior to ETD analysis is evaluated by comparison of three derivatization reagents: iodoacetamide (IAM), N,N-dimethyl-2-chloro-ethylamine (DML), and (3-acrylamidopropyl)-trimethyl ammonium chloride (APTA). While both the DML and APTA modifications lead to an increase in the charge states of peptides, the APTA-peptides provided the most significant improvement in percent fragmentation and sequence coverage for all peptides upon ETD, including formation of diagnostic ions that allow characterization of both the C- and N-termini. In addition, the formation of product ions in multiple charge states upon ETD is minimized for the APTA-modified peptides.
Vasicek
Lisa
L
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, USA.
Brodbelt
Jennifer S
JS
D000478:Alkylation; D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004579:Electron Transport; D005021:Ethylamines / Q000737:chemistry; D007460:Iodoacetamide / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics; D050337:Trimethyl Ammonium Compounds / Q000737:chemistry
D005021:Ethylamines; D010455:Peptides; D050337:Trimethyl Ammonium Compounds; D003545:Cysteine; C041564:ethylamine; D007460:Iodoacetamide