DICP OpenIR
Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome
Chen, Lingfan1,2; Shan, Yichu1; Weng, Yejing1,2; Yuan, Huiming1; Zhang, Shen1,2; Fan, Runlong3; Sui, Zhigang1; Zhang, Xiaodan1; Zhang, Lihua1; Zhang, Yukui1
KeywordC-termini Dimethylation Succinylation Tio2 Particle Proteolysis
Source PublicationANALYTICAL AND BIOANALYTICAL CHEMISTRY
2016-05-01
ISSN1618-2642
DOI10.1007/s00216-016-9476-1
Volume408Issue:14Pages:3867-3874
Indexed BySCI
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS KeywordSUBSTRATE-SPECIFICITY PROFILES ; PROTEIN ; IDENTIFICATION ; ENRICHMENT ; PROTEOMICS ; LIBRARIES ; STRATEGY ; SPECTRA ; RESIN ; MS
AbstractThe analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with alpha-amines and internal peptides bearing both alpha- and epsilon-amines. After the alpha-amines were blocked by site-selective dimethylation or succinylation, the remaining epsilon-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini.
Language英语
WOS IDWOS:000375057400019
PublisherSPRINGER HEIDELBERG
Citation statistics
Document Type期刊论文
Identifierhttp://cas-ir.dicp.ac.cn/handle/321008/170873
Collection中国科学院大连化学物理研究所
Corresponding AuthorZhang, Lihua
Affiliation1.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Liaoning, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Chinese Acad Geol Sci, Beijing SHRIMP Ctr, Inst Geol, Beijing 102206, Peoples R China
Recommended Citation
GB/T 7714
Chen, Lingfan,Shan, Yichu,Weng, Yejing,et al. Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome[J]. ANALYTICAL AND BIOANALYTICAL CHEMISTRY,2016,408(14):3867-3874.
APA Chen, Lingfan.,Shan, Yichu.,Weng, Yejing.,Yuan, Huiming.,Zhang, Shen.,...&Zhang, Yukui.(2016).Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome.ANALYTICAL AND BIOANALYTICAL CHEMISTRY,408(14),3867-3874.
MLA Chen, Lingfan,et al."Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome".ANALYTICAL AND BIOANALYTICAL CHEMISTRY 408.14(2016):3867-3874.
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