Integrated Membrane Proteomic Strategies Revealed Cell Surface Signature during Human Embryonic Stem Cell Differentiation

In stem cell research, cell surface markers are extensively used for stem cell classification, monitoring the differentiation stages as well as purification for their use in regenerative medicine. Quantitative membrane proteomic approaches will provide an in-depth view of the stage- and lineage-specific expression which potentially enhances our understanding on the underlying mechanisms of stem cell differentiation, as well as the opportunity towards isolation of homogenous primary stem cell population. However, the analysis of membrane proteome and its highly heterogeneous glycosylation is experimentally challenging because of their hydrophobic nature and low abundance, which seriously complicate their solubilization, sample handling, separation, and mass spectrometric analysis.
In attempt to search for novel stem cell surface markers and differentiation regulators, we have applied a subglobal proteomic approach and glycoproteomic profiling to define a “membrane proteomic profile” of human embryonic stem (hES) cells and 16-day differentiated embryoid body (EB) outgrowth. Using our recently reported gel-assisted digestion and iTRAQ labeling approach, 3842 proteins were identified (p<0.05) and 2783 proteins were quantified with 2 peptides. By labeling strategy with alkynyl sugar derivatives, the preliminary results In glycoproteomic analysis identified 350 glycopeptides (p<0.05) and quantified in 212 glycoproteins. By combining the quantitative information in protein expression and N-glycosylated peptides, the site-specific glycosylation degree of peptides can be confidently determined on a proteome scale. Our study revealed the dramatic change in expression as well as sialylated N-glycosylation on cell surface glycoproteome during stem cell differentiation. Interestingly, the proteomic data revealed that some cell surface markers, previously discovered by gene expression array, have unaltered expression during stem cell differentiation, which may be due to differences in protein turnover and regulation of the abundance of cellular mRNA and proteins. Mapping of these differentially expressed glycoproteins and membrane proteins in multiple cellular pathways related to cell differentiation, proliferation and cell development suggests that not only the protein markers but also the site and degree of glycosylation have distinct pattern or function in the complex process during stem cell differentiation.