Quantitative Phosphoproteomics Methods Based on Isoelectric Focusing and 18O Labeling Method

Shao Hui SUI, Jing Lan WANG, Wei JIA, Zhuang LU, Jin Feng LIU, Li Na SONG, Yun CAI, Xiao Hong QIAN

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

For the analysis of quantitative phosphoproteomics, both qualitative and quantitative strategies are needed to be studied. Therefore, 18O labeling method was used to label the tryptic phosphopeptides in this study. The labeling condition including the labeling time and the inactivation of the trypsin after labeled were optimized. The experimental results indicated that the incorporation of oxygen-18 isotopes for almost all peptides but the C-terminal peptides not ending on a lysine or an arginine could drive to 100% with tryptic catalyzing in the KH2PO4 buffer system (pH 4-5) for 19-24 h at 37 °C. And tris(2-carboxyethyl)-phosphine chosen as the inactivation of trypsin could effectively inhibit the exchange of incorporated oxygen-18 isotopes with oxygen-16 isotopes. Then, the phosphopeptides enrichment technology IPG-IEF was built, 491 phosphosites, 362 phosphopeptides, and 356 phosphoproteins were identified from HepG2 cells. This suggested that IPG-IEF was effective in the phosphopeptides enrichment analysis on a large scale and could be well compatible with 18O labeling method. Finally, combining with LTQ-FTICR mass spectrometry, 18O-IPG-IEF-LTQ-FTICR was built and demonstrated to be effective in qualitative and quantitative phosphoproteomics study by the experiment results. This study provided useful technology for quantitative phosphoproteome.

Original languageEnglish (US)
Pages (from-to)1017-1023
Number of pages7
JournalChinese Journal of Analytical Chemistry
Volume36
Issue number8
DOIs
StatePublished - Aug 2008
Externally publishedYes

Keywords

  • O labeling method
  • Isoelectric focusing
  • Linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FTICR-MS)
  • Qualitative and quantitative phosphoproteomics

ASJC Scopus subject areas

  • Analytical Chemistry

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