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

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.