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Anal Chem. 2015 Oct 6;87(19):9916-22. doi: 10.1021/acs.analchem.5b02437.

Positive enrichment of C-terminal peptides using oxazolone chemistry and biotinylation.

Author information

1
Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.
2
Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.

Abstract

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.

PMID:
26356223
DOI:
10.1021/acs.analchem.5b02437
[Indexed for MEDLINE]

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