title: Positive enrichment of C-terminal peptides using oxazolone chemistry and biotinylation.
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.
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.
authors.forename: Minbo
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.
authors.forename: Caiyun
authors.initials: C
authors.lastname: Fang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.
authors.forename: Xiuwen
authors.initials: X
authors.lastname: Pan
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.
authors.forename: Hucong
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.|Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University , Shanghai 200032, People's Republic of China.|Department of Chemistry, Fudan University , Shanghai 200433, People's Republic of China.
authors.forename: Haojie
authors.initials: H
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D006736:Horses; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D046968:Thermoanaerobacter / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011506:Proteins; D010081:Oxazolone