Glycoconj J (2012) 29:411–424 DOI 10.1007/s10719-012-9420-3 Surface glycoproteomic analysis of hepatocellular carcinoma cells by affinity enrichment and mass spectrometric identification Wei Mi & Wei Jia & Zhaobin Zheng & Jinglan Wang & Yun Cai & Wantao Ying & Xiaohong Qian Received: 14 April 2012 /Revised: 5 June 2012 /Accepted: 12 June 2012 /Published online: 1 July 2012 # Springer Science+Business Media, LLC 2012 Abstract Cell surface glycoproteins are one of the most surface-capturing (CSC) technique was an approach specif- frequently observed phenomena correlated with malignant ically targeted at membrane glycoproteins involving the growth. Hepatocellular carcinoma (HCC) is one of the most affinity capture of membrane glycoproteins using glycan malignant tumors in the world. The majority of hepatocel- biotinylation labeling on intact cell surfaces. To characterize lular carcinoma cell surface proteins are modified by glyco- the cell surface glycoproteome and probe the mechanism of sylation in the process of tumor invasion and metastasis. tumor invasion and metastasis of HCC, we have modified Therefore, characterization of cell surface glycoproteins can and evaluated the cell surface-capturing strategy, and ap- provide important information for diagnosis and treatment plied it for surface glycoproteomic analysis of hepatocellu- of liver cancer, and also represent a promising source of lar carcinoma cells. In total, 119 glycosylation sites on 116 potential diagnostic biomarkers and therapeutic targets for unique glycopeptides were identified, corresponding to 79 hepatocellular carcinoma. However, cell surface glycopro- different protein species. Of these, 65 (54.6 %) new pre- teins of HCC have been seldom identified by proteomics dicted glycosylation sites were identified that had not pre- approaches because of their hydrophobic nature, poor solu- viously been determined experimentally. Among the bility, and low abundance. The recently developed cell identified glycoproteins, 82 % were classified as membrane proteins by a database search, 68 % had transmembrane domains (TMDs), and 24 % were predicted to contain 2–13 TMDs. Moreover, a total of 26 CD antigens with 50 glyco- Wei Mi and Wei Jia The authors contributed equally to this work. peptides were detected in the membrane glycoproteins of hepatocellular carcinoma cells, comprising 43 % of the total Electronic supplementary material The online version of this article (doi:10.1007/s10719-012-9420-3) contains supplementary material, glycopeptides identified. Many of these identified glycopro- which is available to authorized users. teins are associated with cancer such as CD44, CD147 and EGFR. This is a systematic characterization of cell surface W. Mi : W. Jia : Z. Zheng : J. Wang : Y. Cai : W. Ying (*) : X. Qian (*) glycoproteins of HCC. The membrane glycoproteins identi- State Key Laboratory of Proteomics, Beijing Proteome Research fied in this study provide very useful information for probing Center, Beijing Institute of Radiation Medicine, the mechanism of liver cancer invasion and metastasis. 33 Life Park Road( Changping District( Beijing 102206, ’ People s Republic of China . e-mail: [email protected] Keywords Surface glycoproteins Hepatocellular e-mail: [email protected] carcinoma cells . Mass spectrometry W. Mi Abbreviations Division of Medical and Biological Measurements, National Institute of Metrology, MS mass spectrometry No.18, Bei San Huan Dong Lu, MS/MS tandem mass spectrometry Beijing 100013, People’s Republic of China LC liquid chromatography peptide-N-glycosidase F PNGase F W. Jia Institute of Biophysics, Chinese Academy of Science, TMDs transmembrane domains No. 15 Datun Road( Chaoyang District( Beijing 100101, China GRAVY grand average of hydrophobicity 412 Glycoconj J (2012) 29:411–424 Introduction offers the opportunity for direct labeling and enrichment of membrane glycoproteins on the cell surface [16, 17]. Cell surface membrane proteins straddle the lipid bilayer In this paper, we modified and applied a highly specific boundary between the cell and the environment surrounding and simple approach for surface glycoproteomic analysis of it, and they play many important roles in fundamental bio- the hepatocellular carcinoma cells by targeted enrichment logical processes. Membrane proteins have been extensively and identification of cell membrane glycoproteins. The targeted for drug design, and they account for 70 % of all method involved several steps. First, the cell surface mem- known drug targets [1–3]. One feature of mammalian mem- brane glycoproteins of intact cells were mildly oxidized and brane proteins is that most of them are extensively glycosy- specifically labeled with a biotin group. Second, after cell lated [4, 5]. Glycosylation of membrane proteins plays a key lysis, a simple centrifugation process, protein extraction and role in biological processes such as signal transduction, cell tryptic digestion, the biotinylated membrane glycopeptides adhesion and cell recognition. Some membrane glycopro- were enriched via affinity capture using immobilized avidin. teins have been reported to be involved in cancer progres- Finally, the samples were separated and identified by LC- sion and immune responses [6–8] in carcinomas [9]. MS. The feasibility of the biotin label and capture was Hepatocellular carcinoma (HCC) is one of the most malig- demonstrated by standard glycoproteins. We applied the nant tumors in the world due to high rate of metastasis and strategy to surface glycoproteomic analysis of the hepato- relapse, low rate of 5-year patient survival and poor prog- cellular carcinoma cells. In total, 119 glycosylation sites nosis. Including hepatocellular carcinoma cells, the majority from 116 glycopeptides were identified, corresponding to of the tumor cell membrane proteins are modified by gly- 79 different protein species. Over half (65) of the N- cosylation, but also in the process of tumor invasion and glycosylation sites identified had not been experimentally metastasis with the change of disease process. The adhesion validated in the Uniprot database, with 49 potential glyco- and receptor factors associated with HCC metastasis are sylation sites and 16 unknown sites. Many cell surface mostly membrane glycoproteins [10]. Therefore, proteomic glycoproteins functionally associated with hepatocellular analysis of cell surface glycoproteins for hepatocellular car- carcinoma such as CD44, CD147 were identified. These cinoma cells can provide important information for the early glycoproteins on the cell surface play their roles associated diagnosis of HCC, process monitoring, prognostic assess- with cell adhesion, proliferation, and motility. They are ment and exploring of the therapeutic targets. correlated with tumor prognosis and regulate tumor progres- Many researchers have focused on distinct subsets of sion and metastasis. The strategy and the data were expected membrane glycoproteins. Unfortunately, the purification of to facilitate the researches on membrane glycoproteome and membrane glycoproteins is particularly difficult. Their low biomarker discovery for hepatocellular carcinoma. solubility and low abundance has severely limited the pro- teomic profiling of this class of proteins from complex biological mixtures. The advances in membrane proteomics Materials and methods [11] and glycoproteomics [12] have enabled the study of membrane glycoproteins from a variety of membrane prep- Chemicals and reagents arations with impressive results. Several species of lectins and hydrazine have been used in the second step of the All standard proteins and periodate were obtained from enrichment of cell surface glycoproteins following the initial Sigma-Aldrich (St. Louis, MO). Tris(2-carboxyethyl)phos- step of cell membrane purification by ultracentrifugation phine (TCEP) and EZ-Link Biotin-LC-Hydrazide were [13, 14]. As membranes often lose their specific structure obtained from Pierce (Rockford, US). Protease inhibitor upon cell lysis, a typical membrane-rich fraction prepared cocktail and peptide-N-glycosidase F (PNGase F) were from by ultracentrifugation is heavily contaminated with other Roche (Mannheim, Germany). Cell culture reagents and membrane components from subcellular compartments media were from Hyclone (Logan, UT). Sequence grade [15]. The reliability of the identification results obtained trypsin was from Promega (Madison, WI). All other chem- using this strategy is primarily dependent on the quality of icals were purchased from Thermo Fisher Scientific (Wal- the membrane fraction. Furthermore, the purification proce- tham, MA). dures for cell surface membranes are complicated and time- consuming. Thus, we questioned whether alternative purifi- Cell culture and cell surface labeling cation strategies could be developed that did not rely on membrane purification. As membrane glycoproteins are po- The hepatocellular carcinoma cell line HepG2 was grown at sitioned in the cell membrane with their glycans on the 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) outside of the cell, they are naturally separated from other with 10 % FBS until approaching confluency (~90 %). components of the cell. Thus, the position of these glycans Then, 1×108 cells were washed with PBS (10 mM Glycoconj J (2012) 29:411–424 413 NaH2PO4/Na2HPO4, pH 7.2, 138 mM NaCl, 2.7 mM KCl) was added to dissolve the pellet that was not solubilized by three times, and then incubated in the dark at 4 °C for 8 M urea buffer, and the supernatant was collected