title: [Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins].
abstract: Reduction and alkylation with iodoacetamide (IAA) of the disulfide bridges in proteins are important procedures used in protein digestion. But the alkylation with IAA takes place not only on cysteine residues but also on other amino acid residues. Here we conducted a systematic study of all alkylated peptides under the usual protein digestion conditions. It showed that the potentials for alkylation reaction of different amino acid residues were cysteine > N-terminal amino acid > aspartic acid > glutamic acid > histidine > asparagine > lysine > tyrosine. Furthermore, we found that the alkylation reaction happened either exclusively or cooperatively among different amino acid residues in the same peptide. Based on the qualitative results on overalkylation at several peptides, the targeted multiple reaction monitoring (MRM) technique was used to evaluate the effect of overalkylation on quantitative protein analysis. The results showed that overalkylation has large effect on the qualitative and quantitative analysis of proteins and should be avoided in enzymatic digestion.
authors.affiliation: College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China.
authors.forename: Jifeng
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Ma
authors.affiliation: College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China.
authors.forename: Rugang
authors.initials: R
authors.lastname: Zhong
authors.affiliation: College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
mesh_terms: D000477:Alkylating Agents; D000478:Alkylation; D000596:Amino Acids; D003545:Cysteine; D004220:Disulfides; D007460:Iodoacetamide / Q000737:chemistry*; D010455:Peptides; D011506:Proteins / Q000737:chemistry*
chemical_list: D000477:Alkylating Agents; D000596:Amino Acids; D004220:Disulfides; D010455:Peptides; D011506:Proteins; D003545:Cysteine; D007460:Iodoacetamide