Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential source for treatment of neurological diseases such as Parkinson´s disease. Membrane proteins are very important in cellular signaling and they are regulated by post-translational modifications such as phosphorylation and glycosylation. In order to obtain more information about important membrane proteins and modification sites involved in the differentiation of hESCs to hNSCs and also investigate potential new markers for two stages, we have performed a comprehensive mass-spectrometry-based quantitative proteomics and PTMomics study. Methods: The hESC and hNSC were subject to Na2CO3 and ultracentrifugation to separate the membrane proteins from soluble proteins. The membrane fraction was digested using Lys-C and trypsin, and the resulting peptides were labeled with dimethyl and combined in a 1:1 ratio. Phosphopeptides and sialylated (SA) glycopeptides were enriched using TiO2 followed by SIMAC. SIMAC was performed to separate multi-phosphopeptides from mono-phosphopeptides, SA-glycopeptides and, non-modified peptides. The multiphosphopeptides were analyzed directly by LC-MS/MS. The other peptide fraction was subject to a second TiO2 followed by deglycosylation and HILIC to partly separate mono-phosphopeptides from formerly SA glycopeptides prior to LC-MSMS. Phosphopeptides, former SA glycopeptides and non-modified peptides were identified and quantified; and regulated proteins were further analyzed using bioinformatics and pathway software. Preliminary data: Using our strategy, we were able to identify and quantify 2049 non-modified proteins. Most of them are membrane protein (57%), but surprisingly 43% of proteins did not have transmembrane domains or signal peptides, which suggests either contamination with cytoplasmic proteins or that these proteins can be modified by specific lipids. A total of 5761 phosphorylated peptides mapped to 2011 proteins were identified, which represented 5187 non redundant phosphorylation sites with less than 1% FLR. The 1532 identified former SA glycopeptides from 769 proteins contained 1409 non-redundant glycosylation sites. Membrane proteins represented 50% and 92% of total identified phosphorylated and SA glycosylated proteins, respectively. This study allowed us to identify several significantly regulated proteins during the differentiation process, including proteins involved in the early embryonic development as well as in the neural development. In the latter group of proteins we could identify a number of proteins associated with synaptic vesicles, which are vesicles that store neurotransmitters in the nerve-terminals. An example of an upregulated protein in hESCs is the gap junction alpha 1 (GJA1), a phosphorylated protein which plays a crucial role in embryonic development and has been implicated in cell growth control. Neuronal cell adhesion molecule (NRCAM) is a sialylated glycoprotein upregulated in hNSCs. This protein is involved in several aspects of nervous system development, such as synaptogenesis. In addition, components of neural specific canonical pathways such as cdk5 and neuregulin signalling were found highly upregulated in hNSCs. Moreover, we identified known hNSC markers such as nestin, but also novel potential hNSC markers such as crumbs homolog 2 and protein wntless homolog. These data can be useful to gain a better understanding of the differentiation process and reveal important markers for embryonic and neural development. Novel aspect: First comparative study of the differentiation of hESCs to hNSCs that include quantitative data on phosphorylation and SA glycosylation.
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60th ASMS Conference on Mass Spectrometry and Allied Topics, 2012