Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry

	Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry
	Dai, J; Wang, LS; Wu, YB; Sheng, QH; Wu, JR; Shieh, CH; Zeng, R
刊名	JOURNAL OF PROTEOME RESEARCH
	2009
卷号	8 期号:1 页码:133-141
关键词	Phosphoproteomics Phosphopeptides Two-dimensional Anion exchange chromatography pH continuous online gradient Mass spectrometry
通讯作者	Shieh, CH (reprint author), Chinese Acad Sci, Key Lab Syst Biol, Inst Biochem & Cell Biol, Shanghai Inst Biol Sci, Shanghai 200031, Peoples R China.,jhxie@sibs.ac.cn ; zr@sibs.ac.cn
英文摘要	Most current technologies for the enrichment of phosphopeptides rely on a tandem combination of different chromatography modes. Here, a fully automatic two-dimensional liquid chromatography mass spectrometry method was developed for global phosphopeptide identification. The peptide mixtures were loaded on a strong anion exchange (SAX) column under basic pH conditions and eluted with a continuous gradient to pH 2.0. This SAX system could be coupled online with reversed-phase liquid chromatography mass spectrometry (RP-LC-MS/MS). For peptide digests from a standard protein mixture spiked with synthesized phosphopeptides, most of the nonphosphorylated peptides were eluted in more basic pH than phosphopeptides, and the phosphopeptides were focused to acidic pH ranges and gradually eluted according to the phosphorylated states of peptides. Compared with the pH step elution method, the continuous gradient method displayed better repeatability and less peptide cross-overlap between fractions. This system provided a robust and fully automatic approach to large-scale phosphoproteomic profiling. For protein tryptic digests from HeLa cells, 1833 nonredundant phosphorylation sites were identified based on this two-phase separation. Compared with the method combining cation exchange and titanium dioxide, this anion-exchange based system preferred to identify more acidic and multi phosphorylated peptides. It also covered a more complete series of phosphorylation states of peptides.
学科主题	Biochemistry & Molecular Biology
类目[WOS]	Biochemical Research Methods
关键词[WOS]	METAL-AFFINITY-CHROMATOGRAPHY ; PROTEIN-PHOSPHORYLATION ; PHOSPHOPROTEOME ANALYSIS ; SELECTIVE ENRICHMENT ; SIGNALING NETWORKS ; ION-EXCHANGE ; MOUSE ; FRACTIONATION ; PEPTIDES ; KINASES
收录类别	SCI
语种	英语
WOS记录号	WOS:000262171100015
内容类型	期刊论文
版本	出版稿
源URL	[http://202.127.25.143/handle/331003/1135]
专题	上海生化细胞研究所_上海生科院生化细胞研究所
推荐引用方式 GB/T 7714	Dai, J,Wang, LS,Wu, YB,et al. Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry[J]. JOURNAL OF PROTEOME RESEARCH,2009,8(1):133-141.
APA	Dai, J.,Wang, LS.,Wu, YB.,Sheng, QH.,Wu, JR.,...&Zeng, R.(2009).Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry.JOURNAL OF PROTEOME RESEARCH,8(1),133-141.
MLA	Dai, J,et al."Fully Automatic Separation and Identification of Phosphopeptides by Continuous pH-Gradient Anion Exchange Online Coupled with Reversed-Phase Liquid Chromatography Mass Spectrometry".JOURNAL OF PROTEOME RESEARCH 8.1(2009):133-141.