In Lesch-Nyhan syndrome, the impaired purinergic SJN 2511 ALK inhibitor metabolism is initiated from cell-surface purinergic receptors which are also modified by N-glycans. Hence, decoding the N-glycoproteome of E14.Tg2a will benefit the research and application of mES cells, as well as the studies of the pathophysiology of Lesch-Nyhan syndrome. A comprehensive characterization of the N-glycoproteome is, SCH772984 however, technically challenging. membrane-bound N-glycoproteins inherit the challenges of membrane-protein studies: the low solubility in aqueous solution and the low abundance. To conquer these challenges, researchers have separated glycancentric glycomics from protein-centric glycoproteomics in highthroughput analyses. For protein-centric N-glycoproteomics, the enrichment of N-glycoproteins is often necessary for sensitive analyses, and several techniques have been developed for this purpose, including the lectin affinity enrichment, boronic acid and hydrazide based chemical enrichments among other chemical or physical methods. To date, N-glycoproteomics has been carried out to mES cells, yet no effort has been focused on the E14.Tg2a subclone. To characterize the N-glycoproteome of E14.Tg2a, we used a previously developed N-glycopeptide capture strategy, a technique optimized for membrane N-glycoproteins. Using conventional liquid chromatography and low-end mass spectrometer, we cataloged the low-abundance N-glycoproteins and their glycosylation patterns in E14.Tg2a cells. The subsequent data analyses allowed us to discover some novel structural and functional relations among membrane proteins. All proteomic data presented here has been deposited in the publically available Peptide Atlas database. Stem cells possess unique properties and broad medical applications. Understanding and harnessing these almighty cells have a profound impact in developmental biology, regenerative medicine and personalized therapies. Membrane proteins help mediate the communication between stem cells and their immediate environments, thereby playing pivotal roles in stem-cell biology. The characterization of membrane proteins allows functional understanding and a potential control of these molecules. In addition, a proteomic-scale analysis enables holistic views of the cell surface, in which different yet simultaneously happening processes and functions can be examined together and novel synergies can be discovered. Even though large-scale genomic analyses have greatly advanced our understanding of stem cells, direct protein analyses are necessary and important for numerous reasons. One of the important reasons is that many protein functions are regulated by post-translational processes, which are beyond the scope of genomics. We therefore used proteomic approach and characterized in the current study proteins with N-glycosylation, one of the most structurally complex co- and post-translational modifications in biological systems. The unique cellular localization of N-glycoproteins allowed us to effectively identify cell surface proteins when the results were compared with other membrane proteomes. In addition, our results described the sites and stoichiometry of protein N-glycosylation at proteomic scale, enabling the building of novel links between protein structure and function that genomic studies cannot provide.