Microamount Phosphopeptide-Enrichment-System-Based Phosphoproteomic Analysis for Small Numbers of Cells and Single...

Protein phosphorylation plays an important role in cellular regulation and signal transduction. In this study, we developed a microamount phosphopeptide enrichment system (mPES) to achieve the enrichment of phosphopeptides in nanoliter-scale based on microfluidics and the solid-phase microextraction (SPME) technique. Based on mPES, we established a complete set of phosphoproteomic analysis workflow for trace cell samples, including operations of cell capture, cell lysis, protein proteolysis, phosphopeptide enrichment, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, which can profile phosphoproteomics of complex samples as low as picogram amounts. Cells pretreatment and phosphopeptide enrichment were carried out in the nanoliter-scale volume range, which significantly increased the enrichment efficiency and reduced the sample loss by container adsorption. We applied the present system to the analysis of phosphorylation in 1, 3, 5, 10 HeLa cells, and 83, 117, 187, 227 phosphopeptides were identified after enrichment, respectively. The intensities of phosphopeptides increased by 2.91, 2.80, 8.81, and 9.95 times compared with the control groups. The mPES was further applied to the phosphoproteomic analysis of single mouse oocytes, with an average of 221 phosphoproteins identified in single germinal vesicle (GV) oocytes and 460 in single metaphase II (MII) oocytes, which provided a single-cell perspective for further elucidating the mechanism of oocyte maturation.