Positive Enrichment of C-Terminal Peptides Using Oxazolone Chemistry and Biotinylation
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- 作者：Minbo Liu ; Caiyun Fang ; Xiuwen Pan ; Hucong Jiang ; Lijuan Zhang ; Lei Zhang ; Ying Zhang ; Pengyuan Yang ; Haojie Lu
- 刊名：Analytical Chemistry
- 出版年：2015
- 出版时间：October 6, 2015
- 年：2015
- 卷：87
- 期：19
- 页码：9916-9922
- 全文大小：359K
- ISSN：1520-6882
文摘
Selective capture of protein C-termini is still challenging in view of the lower reactivity of the carboxyl group relative to amino groups and difficulties in site-specifically labeling the carboxyl group on the C-terminus rather than that on the side chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with a streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by mass spectrometry (MS). Unlike the negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from the peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio), and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing the database search strategy. We have successfully identified 183 C-terminal peptides from Thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.