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学科主题: 物理化学
题名: Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment
作者: Zhou, Houjiang1, 2, 3;  Low, Teck Y.1, 2, 3;  Hennrich, Marco L.1, 2, 3;  van der Toorn, Henk1, 2, 3;  Schwend, Thomas1, 2, 3;  Zou, Hanfa4;  Mohammed, Shabaz1, 2, 3;  Heck, Albert J. R.1, 2, 3
通讯作者: AlbertJ.R.Heck
刊名: MOLECULAR & CELLULAR PROTEOMICS
发表日期: 2011-10-01
DOI: 10.1074/mcp.M110.006452
卷: 10, 期:10, 页:1
收录类别: SCI
文章类型: Article
部门归属: 18
项目归属: 1809
产权排名: 3,6
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemical Research Methods
摘要: Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment
研究领域[WOS]: Biochemistry & Molecular Biology
英文摘要: Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti4+-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO2 enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti4+-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 mu g of a HeLa cell lysate digest. In comparison, similar to 2000 unique phosphopeptides could be identified by Ti4+-IMAC with HFP and close to 3000 by TiO2. We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti4+-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 mu g of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti4+-IMAC is particularly advantageous for phosphopeptides with multiple basic residues. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006452, 1-14, 2011.
关键词[WOS]: ELECTRON-TRANSFER DISSOCIATION ;  TANDEM MASS-SPECTROMETRY ;  HYDROPHILIC INTERACTION CHROMATOGRAPHY ;  ION AFFINITY-CHROMATOGRAPHY ;  PHOSPHOPROTEOME ANALYSIS ;  PHOSPHORYLATED PEPTIDES ;  PROTEIN-PHOSPHORYLATION ;  IN-VIVO ;  QUANTITATIVE PHOSPHOPROTEOMICS ;  SACCHAROMYCES-CEREVISIAE
语种: 英语
WOS记录号: WOS:000295773800026
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/115298
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Univ Utrecht, Biomol Mass Spectrometry & Prote Grp, Bijvoet Ctr Biomol Res, NL-3584 CH Utrecht, Netherlands
2.Univ Utrecht, Utrecht Inst Pharmaceut Sci, NL-3584 CH Utrecht, Netherlands
3.Netherlands Prote Ctr, NL-3584 CH Utrecht, Netherlands
4.Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China

Recommended Citation:
Zhou, Houjiang,Low, Teck Y.,Hennrich, Marco L.,et al. Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment[J]. MOLECULAR & CELLULAR PROTEOMICS,2011,10(10):1.
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