High throughput functional proteomics for surface proteins on mouse embryonic stem cells
Proteomics is a high throughput analysis of the structure and function of proteins in biological systems. Our glycoprotein-targeted proteomic analysis employs liquid chromatography (LC) as a separation tool in line with mass spectrometry (MS) as an analyser to identify and quantify glycoproteins and their site of glycosylation. In a typical analysis, proteins will be extracted from biological samples, digested into peptides enzymatically, and glycosylated peptides will be chemically enriched. The N-linked glycopeptides will be selectively analyzed through LC-MS. Thousands of peptide MS spectra can be acquired in a short duration and processed by bioinformatics tools to infer protein structure and function.
This project will focus on improving the sensitivity of glycoproteomics method that we have developed previously (Sun, et. al., MCP, 2007). Successful remedies will be deployed to study membrane proteins of mES cells. Our lab has extensive experiences with these cells, and we have both host and gene knockout mES cells in culture. The new knowledge gained from these newly developed sensitive analyses will be evaluated easily through comparison to our curated databases in identifying stem-cell specific protein markers. Connections drawn between surface molecular topology and intracellular protein networks could also provide us novel insights into stem cell biology and physiology.
Exicuting such project will provide students hands on experiences on all aspects of proteomics technology, including protein chemistry, liquid chromatography separation techniques, tandem mass spectrometry, and data-analysis skills. Our lab also harbours various molecular and cellular techniques, such as mammalian cell culture and assays, immunoassays, fluorescence imaging, and single-cell assays, which can be acquired during training. Successfully trained students will have opportunity to continue the project as a graduate student.
