Citation: WANG Jifeng, ZHAO Xinyuan, ZHAO Yan, MA Cheng, ZHONG Rugang, QIAN Xiaohong, YING Wantao. Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins[J]. Chinese Journal of Chromatography, ;2013, 31(10): 927-933. doi: 10.3724/SP.J.1123.2013.04041 shu

Influence of overalkylation in enzymatic digestion on the qualitative and quantitative analysis of proteins

WANG Jifeng ^1,2 ,
ZHAO Xinyuan ^1,2 ,
ZHAO Yan ² ,
MA Cheng ² ,
ZHONG Rugang ¹ ,
QIAN Xiaohong ^1,2 ,
YING Wantao ^2,,

1.
1. College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100022, China;

Corresponding author: YING Wantao,
Received Date: 23 April 2013
Available Online: 13 May 2013
Fund Project: 国家重点基础研究"973"项目(2012CB910301) (2012CB910301)国家高技术研究发展计划项目(2012AA020201) (2012AA020201)国家自然科学基金项目(31100591). (31100591)

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.
- liquid chromatography (LC),
- mass spectrometry (MS),
- multiple reaction monitoring (MRM),
- proteomics,
- alkylation,
- iodoacetamide
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