N-Glycoproteome of E14.Tg2a Mouse Embryonic Stem Cells Bingyun Sun1,2*,LiMa1, Xiaowei Yan1, Denis Lee1, Vinita Alexander1, Laura J. Hohmann1, Cynthia Lorang1, Lalangi Chandrasena2, Qiang Tian1, Leroy Hood1* 1 Institute for Systems Biology, Seattle, Washington, United States of America, 2 Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada Abstract E14.Tg2a mouse embryonic stem (mES) cells are a widely used host in gene trap and gene targeting techniques. Molecular characterization of host cells will provide background information for a better understanding of functions of the knockout genes. Using a highly selective glycopeptide-capture approach but ordinary liquid chromatography coupled mass spectrometry (LC-MS), we characterized the N-glycoproteins of E14.Tg2a cells and analyzed the close relationship between the obtained N-glycoproteome and cell-surface proteomes. Our results provide a global view of cell surface protein molecular properties, in which receptors seem to be much more diverse but lower in abundance than transporters on average. In addition, our results provide a systematic view of the E14.Tg2a N-glycosylation, from which we discovered some striking patterns, including an evolutionarily preserved and maybe functionally selected complementarity between N- glycosylation and the transmembrane structure in protein sequences. We also observed an environmentally influenced N- glycosylation pattern among glycoenzymes and extracellular matrix proteins. We hope that the acquired information enhances our molecular understanding of mES E14.Tg2a as well as the biological roles played by N-glycosylation in cell biology in general. Citation: Sun B, Ma L, Yan X, Lee D, Alexander V, et al. (2013) N-Glycoproteome of E14.Tg2a Mouse Embryonic Stem Cells. PLoS ONE 8(2): e55722. doi:10.1371/ journal.pone.0055722 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received September 27, 2012; Accepted December 29, 2012; Published February 6, 2013 Copyright: ß 2013 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This publication was made possible by Grant Numbers 1U54DA021519 and 1U54CA119347 from NIH (National Institutes of Health), 1P50GM076547 from NIH/NIGMS (National Institute of General Medical Sciences), P01 DK53074 from NIH/NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases), and LC is supported by the Startup fund from Simon Fraser University. The contents here are solely the responsibility of the authors and do not necessarily represent the official views of the NIH and Simon Fraser University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (LH); [email protected] (BS) Introduction Lesch-Nyhan syndrome is a metabolic disorder hallmarked by hyperuricemia, mental retardation and self-mutilation [6]. The Embryonic stem (ES) cells exhibit unique properties of self- pathology of the E14.Tg2a host potentially complicates the use of renewal and pluripotency, possessing broad applications in this system in deciphering target gene functions. Therefore, there developmental biology and regenerative medicine [1,2]. Mouse is a need to elucidate molecular details of this cell line itself in ES (mES) cells are valuable tools to produce genetically modified building a well-understood genomic background. Even though mouse strains through gene targeting and gene trapping several high-throughput molecular characterizations have been techniques for studies in functional genomics and biomedical carried out to E14 mES cells, little attention has been directed research. Molecular characterization of host mES cells provides towards the E14.Tg2a subclone (except for the proteomic background information for a better functional understanding of characterization of the chromatin remodeling complex conducted the knockout gene(s). Towards this end, we here focus on by Ho L. et al. in 2009) [7]. Seegmiller et al. stated in 1967 [6] that deciphering the N-glycoproteome of E14.Tg2a, one of the most Lesch-Nyhan syndrome is the first example of an abnormal popular host cell lines used for gene targeting and gene trapping compulsive behavior raised by a specific enzymatic defect; and it is [3]. also the first demonstrated enzymatic defect in purine metabolism The mES cell line, E14.Tg2a, derived by Hooper et al. from in a neurological disease. Thus, it is also interesting to elucidate the 129/OLA in 1987 [4] was originally developed as a mouse model protein makeup of E14.Tg2a mES cells from pathologic and of Lesch-Nyhan disorder with a deficiency of hypoxanthine metabolic viewpoints. phosphoribosyltransferase (HPRT). E14.Tg2a cells grow fast and In stem cells, the choice of proliferation and differentiation is steadily in both feeder and feeder-free systems, and produce higher largely regulated by the interaction between cell surface proteins success of germ-line transmitting chimera than mES cells derived and cells’ microenvironment, i.e. the stem-cell niche. Both the cell from BL6 strains [5]. Therefore, E14.Tg2a is an ideal system for surface and the niche are rich in glycoproteins, especially N-linked genetic engineering. To date, at least 29,000 transgenic mice and glycoproteins. N-glycosylation is a co-translational modification mES cell lines have been derived from E14.Tg2a (Dr. Richard that takes place at the ER, and functions importantly in protein Baldarelli, Mouse Genome Informatics, The Jackson Laboratory, folding, stabilization, membrane trafficking, and interaction with personal communication). other molecules. The complete removal of N-glycosylation from all PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e55722 N-Glycoproteome of E14.Tg2a mES Cells cellular proteins is embryonically lethal [8,9], and the aberrant N- tissue culture dishes in GMEM medium supplemented with 2-mM glycosylation on individual proteins can cause severe birth defects, glutamine, 1-mM sodium pyruvate, nonessential amino acids, including but not limited to the congenital disorder of glycosyl- 10% fetal bovine serum, 50-mM ß-mercaptoethanol, and 1000 U/ ation (CDG) as well as lysosomal storage diseases [10]. N-linked ml leukocyte inhibitory factor. glycoproteins reside specifically at the outer plasma membrane, in The crude membrane fraction of ES cells was prepared the extracellular milieu, secretory channel (i.e. ER and Golgi according to previous procedures [25]. In brief, cultured cells apparatus) and endocytic pathway (lysosomes and endosomes) were washed with phosphate buffered saline (PBS) and removed [11]. The external facing of N-linked polypeptides at the cell-outer from culture dishes by scraping. After centrifugation at 1,500 rpm membrane makes these proteins ideal candidates as markers of for 5 min at 4uC, cells were re-suspended in 10-ml hypotonic stem cells, and most known ES-cell surface markers are indeed N- buffer (20-mM Tris-HCl pH 7.4, 10-mM MgCl2, 10-mM CaCl2) glycoproteins including Thy1 (CD90), c-kit (CD117), Lrp2 containing protease inhibitors, and incubated on ice for 15 min (endoglin), Prom 1 (CD133) and neural cell adhesion molecule prior to homogenization. Cells were lysed by Dounce homogeni- (NCAM) [12,13]. In Lesch-Nyhan syndrome, the impaired zation (,50 strokes), and the lysis was confirmed by trypan-blue purinergic metabolism is initiated from cell-surface purinergic staining. The microsomal fraction was obtained by differential receptors which are also modified by N-glycans [14]. Hence, centrifugation first at 4uC and 3,000 g for 15 min, and then at 4uC decoding the N-glycoproteome of E14.Tg2a will benefit the and 100,000 g for 2 h (Beckman Coulter L8-70M Ultracentrifuge). research and application of mES cells, as well as the studies of the The final pellet containing the plasma membrane and endomem- pathophysiology of Lesch-Nyhan syndrome. brane systems was stored at 280uC. A comprehensive characterization of the N-glycoproteome is, however, technically challenging. N-glycans comprise the most Glycopeptide Capture complex and diversified structures among all known protein post- The microsomal fraction was dissolved in a denaturing buffer translational modifications; and membrane-bound N-glycopro- containing 0.5% RapiGestH and 8-M urea, and digested into teins inherit the challenges of membrane-protein studies: the low peptides prior to glycopeptide capture using a previously described solubility in aqueous solution and the low abundance [15,16]. To protocol [23] with minor modifications. In a typical preparation, conquer these challenges, researchers have separated glycan- about 0.5–0.8 mg microsomal protein was obtained from ,36108 centric glycomics from protein-centric glycoproteomics in high- cells. Proteins were denatured, alkylated, and the sample solution throughput analyses [17–20]. For protein-centric N-glycoproteo- was diluted at least ten times before the trypsin digestion step. The mics, the enrichment of N-glycoproteins is often necessary for detergent, RapiGest, was removed by degrading the digest at pH sensitive analyses, and several techniques have been developed for ,1 for 1 h at 37uC, and the developed precipitation was removed