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Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study
Xu, Feng1; Yu, Li2,3; Peng, Xuehui1,4; Zhang, Junling1; Li, Suzhen1,5; Liu, Shu1; Yin, Yanan4; An, Zhiwu2,3; Wang, Fuqiang1; Fu, Yan2,3; Xu, Ping1,4,5
2020-06-05
Source PublicationJOURNAL OF PROTEOME RESEARCH
ISSN1535-3893
Volume19Issue:6Pages:2185-2194
AbstractUnderstanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side. We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS(2 )spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score >= 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).
Keywordtrypsin LysargiNase combined spectra ion coverage phosphosite localization PTMiner
DOI10.1021/acs.jproteome.9b00562
Indexed BySCI
Language英语
Funding ProjectMOST[2017YFC0906600] ; MOST[2016YFA0501300] ; MOST[2017YFA0505000] ; MOST[2017YFA0505100] ; National Natural Science Foundation of China[31670834] ; National Natural Science Foundation of China[31700723] ; National Natural Science Foundation of China[91839302] ; National Natural Science Foundation of China[31870824] ; Innovation Foundation of Medicine[16CXZ027] ; Innovation Foundation of Medicine[BWS17J032] ; Innovation Foundation of Medicine[BWS14J052] ; National Megaprojects for Key Infectious Diseases[2018ZX10302302001] ; Guangzhou Science and Technology Innovation & Development Project[201802020016] ; Unilevel 21st Century Toxicity Program[MA-2018-02170N] ; Foundation of State Key Lab of Proteomics[SKLP-Y201501] ; Foundation of State Key Lab of Proteomics[SKLP-K201705] ; NCMIS CAS
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemical Research Methods
WOS IDWOS:000538772300002
PublisherAMER CHEMICAL SOC
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Document Type期刊论文
Identifierhttp://ir.amss.ac.cn/handle/2S8OKBNM/51648
Collection中国科学院数学与系统科学研究院
Corresponding AuthorXu, Feng; Fu, Yan; Xu, Ping
Affiliation1.Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China
2.Chinese Acad Sci, Acad Math & Syst Sci, Natl Ctr Math & Interdisciplinary Sci, Key Lab Random Complex Struct & Data Sci, Beijing 100864, Peoples R China
3.Univ Chinese Acad Sci, Sch Math Sci, Beijing 100049, Peoples R China
4.Wuhan Univ, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery, Sch Med,Minist Educ, Wuhan 430072, Peoples R China
5.Anhui Med Univ, Hefei 230032, Peoples R China
Recommended Citation
GB/T 7714
Xu, Feng,Yu, Li,Peng, Xuehui,et al. Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study[J]. JOURNAL OF PROTEOME RESEARCH,2020,19(6):2185-2194.
APA Xu, Feng.,Yu, Li.,Peng, Xuehui.,Zhang, Junling.,Li, Suzhen.,...&Xu, Ping.(2020).Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study.JOURNAL OF PROTEOME RESEARCH,19(6),2185-2194.
MLA Xu, Feng,et al."Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study".JOURNAL OF PROTEOME RESEARCH 19.6(2020):2185-2194.
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