ORIGINAL PAPER

Cell surface glycoprotein profiling of cancer cells basedon bioorthogonal chemistry

Peng-wei Pan & Qi Zhang & Jie Hou & Ze Liu & Fang Bai &Mei-rong Cao & Ting Sun & Gang Bai

Received: 29 November 2011 /Revised: 9 March 2012 /Accepted: 27 March 2012 /Published online: 18 April 2012# Springer-Verlag 2012

Abstract Bioorthogonal chemistry refers to chemicalreactions that can occur within a living system withoutaltering native biochemical processes. Applications of thisconcept extend to studies on a group of biomolecules thatincludes glycans, proteins, and lipids. In this study, astrategy for isolating cell surface glycoproteins and basedon bioorthogonal chemistry was employed to identify newcancer-related glycoproteins. A novel alkyne reagent con-taining one disulfide bond was synthesized for the enrich-ment of glycoproteins metabolized with peracetylatedN-azidoacetylmannosamine, which was applied on threedifferent cancer cell lines, and all isolated proteins wereanalyzed by high-performance liquid chromatography–tandem mass spectrometry. The strategy of purifying cellsurface glycoproteins introduced in this article was shownto be reliable, and a total of 56 cell surface glycoproteinswere identified. Neuronal cell adhesion molecule wasfound uniquely expressed in A549 lung adenocarcinoma,

and its expression in non-small-cell lung carcinomas wasdetected by immunohistochemistry. Furthermore, a signi-ficant increase of neuronal cell adhesion molecule expres-sion was identified in non-small-cell lung adenocarcinomacompared with adjacent noncancerous tissues, and couldbe a novel potential target and marker in cancer treatmentand detection.

Keywords Click chemistry . Sialic acid . Glycoprotein .

Lung adenocarcinoma . Neuronal cell adhesion molecule

Introduction

Glycosylation is an important posttranslational modificationof eukaryotic proteins [1]. This modification plays crucialroles in various biological events, such as cell recognition,inter- and intracellular signaling, cell adhesion, and cell–cellinteraction [2]. Different levels of glycosylation are oftenobserved in pathological cells compared with normal cellsand are regarded as a hallmark of disease states [3].

To investigate glycoprotein expression, several purifica-tion methods that target different typical structures or sub-units have been reported. The hydrazide chemistry methodis generally performed by oxidizing sugar residues, fol-lowed by covalent coupling to hydrazide resin to captureglycosylated peptides [4]. Lectins have an affinity towardsglycolipids and glycoproteins because of their various car-bohydrate moieties. This characteristic has been widely usedin affinity chromatography to purify glycoproteins withcertain glycoforms and carbohydrate structures [5]. Com-bined use of lectins provides a much broader spectrum ofglycoprotein recognition [6]. These two methods are pres-ently the most widely used approaches in studies involving

Electronic supplementary material The online version of this article(doi:10.1007/s00216-012-5989-4) contains supplementary material,which is available to authorized users.

P.-w. Pan : T. SunCollege of Pharmacy and College of Life Sciences,Nankai University,Tianjin 300071, China

Q. Zhang (*) : J. Hou : F. Bai :M.-r. Cao :G. Bai (*)State Key Laboratory of Medicinal Chemical Biology and Collegeof Pharmacy, Nankai University,Tianjin 300071, Chinae-mail: qizhang@nankai.edu.cne-mail: gangbai@nankai.edu.cn

Z. LiuCollege of Medicine, Nankai University,Tianjin 300071, China

Anal Bioanal Chem (2012) 403:1661–1670DOI 10.1007/s00216-012-5989-4

glycoproteins and glycoproteomics. Other methods, such asboronate affinity chromatography [7], the beta eliminationof O-GlcNAc [8], titanium dioxide [9], and hydrophilicinteraction chromatography [10], have also been introducedfor glycoprotein and glycopeptide enrichment.

In addition to the studies mentioned above, a strategyknown as “bioorthogonal chemistry,” which is performed byincorporating detectable chemical motifs into glycan chains,has recently emerged and has received favorable attention.Nonnatural monosaccharides are utilized by the cell’s ownmetabolic system and are incorporated in place of normalmonosaccharides. Chemical tags can then be utilized forisolations or for visualizations. These artificial substitutesshould be stable, nontoxic, and well tolerated by cellularsynthesis mechanisms, and their incorporation into glycanstructures should not alter the cell’s viability [11]. The azidogroup has been found to be an ideal group, as it is reactivewith alkynes and meets these criteria. These types of reac-tions are considered nonnative, nonperturbing, and highlyselective [11, 12].

Sialic acid is typically the outermost monosaccharide uniton the glycan chains of glycolipids and glycoproteins. Saxonand Bertozzi [13] developed a strategy that incorporatedazides on sialic acids within glycoconjugates by culturingcells with peracetylated N-azidoacetylmannosamine(Ac4ManNAz), a peracetylated analog of ManNAc modifiedwith azide (Fig. 1a). Peracetylation can greatly enhance the

uptake and anabolic utilization of carbohydrates, and increasesthe presence of azide groups on glycoproteins [14]. Theazido groups present in cells can be detected using analkyne probe containing various tags in a Cu(I)-catalyzed[3+2] cycloaddition reaction (Fig. 1b) [15]. This strategy hasbeen validated and used in a number of in vitro and in vivoexperiments [16–18].

In our study, similar to the method described by Saxonand Bertozzi, Ac4ManNAz was introduced to label glyco-proteins. A releasable alkyne probe was synthesized andapplied for the comprehensive profiling and identificationof cancer-associated cell surface glycoproteins. Glycopro-teins from three different adenocarcinoma cell lines—A549lung adenocarcinoma, HeLa cervical carcinoma, andSW1990 pancreatic adenocarcinoma—were enriched. Weused liquid chromatography–tandem mass spectrometry(MS) to characterize the proteins obtained. As a result, 310proteins were identified and 56 of these proteins were pre-dicted to be cell surface glycoproteins.

Unique expression patterns of neuronal cell adhesionmolecule (NrCAM) and contactin 1 (CNTN1) were ob-served in A549 lung adenocarcinoma. Both NrCAM andCNTN1 belong to the immunoglobulin-like cell adhesionmolecule family, which might induce the adhesion andmetastasis of tumor cells. Currently, NrCAM has not beenreported to be expressed in non-small-cell lung carcinoma(NSCLC). Immunohistochemistry revealed a significant in-crease in NrCAM expression in non-small-cell lung adeno-carcinoma tissues compared with adjacent noncanceroustissues; however, this increase was not detected in NSCLCsquamous cells. This result indicated that NrCAM mayplay some role in non-small-cell lung adenocarcinomacells and might serve as a novel potential marker in cancerdetection and treatment.

Materials and methods

Compound synthesis

Ac4ManNAz synthesis was performed following the proto-col described by Sampathkumar [19] (see the electronicsupplementary material) [19]. Biotin-LC-alkyne has beendescribed as a probe to detect azido-modified glycoconju-gates [16]. In this study, biotin-LC-alkyne analogs weresynthesized using the modified commercialized reagentssulfo-NHS-LC-biotin and sulfo-NHS-SS-biotin (see theelectronic supplementary material), which can efficientlyreact with primary amino groups to form stable amidebonds. Propargylamine was reacted with sulfo-NHS-SS-biotin or sulfo-NHS-LC-biotin in borate buffer (0.2 M, pH8) to modify one end of the long chain with an alkyne, asshown in Fig. 1c.

Fig. 1 Azido-labeled mannose is metabolized by the cell and ispresent on cell surfaces as SiaNAz (a), which can be detected byalkyne probes through a Cu(I)-catalyzed [3+2] cycloaddition reaction(b). Commercial biotinylation reagents were modified with alkyne bythe reaction of the N-hydroxysulfosuccinimide (NHS) ester group withthe ε-amine of propargylamine lysine residues (c)

1662 P. Pan et al.

Biotinylation and the fluorescent labeling of cell surfaceglycoproteins

Different cell lines were cultured at 37 °C in Dulbecco’smodified Eagle’s medium with 10 % fetal bovine serum,100 U/mL penicillin, 100 ng/mL streptomycin, and 0.2 mMAc4ManNAz or peracetylated N-acetylmannosamine(Ac4ManNAc) for 48 h (HeLa, A549, and SW1990 cellswere obtained from the ATCC, USA). Cells were washedwith ice-cold phosphate-buffered saline (PBS; 0.1 M, pH7.4) and treated with a 100-μL reaction solution (0.2 mMbiotin-SS-alkyne or biotin-LC-alkyne, 0.2 mM tristriazole-amine, 1.0 mM CuSO4, and 2.0 mM sodium ascorbate inice-cold PBS). After 1 h of incubation, the cells werewashed with PBS, fixed in 4 % paraformaldehyde, and thenpermeabilized with 1 % Triton X-100 for 30 min. Avidin–Cy3 was used to stain the cells. After the fluorescent label-ing, 100 mM dithiothreitol (DTT) was added to detectwhether the biotin was released or not. All the fluorescenceimages were obtained by confocal microscopy (TCS SP5,Leica, Germany).

Purification of cell surface glycoproteins

Cells were cultured in Ac4ManNAz as described in theprevious section. After they had been washed with ice-coldPBS, the cells were treated with biotin-SS-alkyne (4 °C). Aftera 1-h incubation period, the reaction solution was removed,and the cells were carefully washed with ice-cold PBS. Thecells were scraped into ice-cold hypotonic buffer [10 mMN-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (pH 7.5),1.5 mM MgCl2, 10 mM KCl, 1× protease inhibitor cocktail,1 mM NaF, and 1 mM Na3VO4] and incubated on ice for15 min, after which they were lysed by sonication. Aftercentrifugation at 1,000g for 10 min (4 °C), the supernatantwas collected, and the KCl concentration was adjusted to150 mM. Typically, 1 mL of suspended streptavidin magneticbeads (10 mg of beads per milliliter, prewashed four timeswith ice-cold hypotonic buffer) was added to 10 mL of lysate(1 mg/mL), and the resulting suspension was shaken at 4 °Cfor 1 h. The beads were collected via a magnetic collector andsequentially washed three times with ice-cold PBS and 1 MKCl. The glycoproteins were released by washing them with100 mM DTT and were quantified using a Bradford proteinassay kit (Solarbio, China).

Protein digestion

For the in-solution digestion of the protein lysate, the pro-teins were reduced in 10 mM DTT, alkylated with 50 mMiodoacetamide in the dark, and digested with trypsin (1:50,w/w) (Promega, USA) at 37 °C for 16 h. Trifluoroacetic acidwas added to block the digestion.

High performance liquid chromatography-MS/MS

The peptides were desalted on RP trap columns (Zorbax 300SB C18, Agilent Technologies, USA) and then separated onan RP column (150-μm inner diameter, 100-mm length,Column Technology, Fremont, CA, USA). A FinniganLTQ linear ion trap mass spectrometer (Thermo Electron,USA) equipped with an electrospray interface wasconnected to the liquid chromatography system for detect-ing the eluted peptides. Data-dependent MS/MS spectrawere simultaneously obtained. Each scan cycle consistedof one full MS scan in profile mode followed by five MS/MS scans in centroid mode, with the following dynamicexclusion settings: repeat count 2, repeat duration 30 s,exclusion duration 90 s. Each sample was analyzed intriplicate.

Data analysis

MS/MS spectra were automatically searched against thenonredundant International Protein Index (IPI) human pro-tein database (version 3.26, 67,687 entries) using Bioworksbrowser version 3.1 (Thermo Electron, USA). Protein iden-tification results were extracted from SEQUEST files usingBuildSummary [20]. The mass tolerances allowed for in theprecursor and fragment ions were 2.0 and 0.8 Da, respec-tively. The protein identification criteria were based onDelta CN (≥0.1) and cross-correlation scores (one charge1.9 or greater, two charges 2.2 or greater, three charges 3.75or greater).

The Gene Ontology database and CBS Predictiontools were used to predict cell surface glycoproteins.First, proteins located in the membrane were analyzedusing STRAP [21]. As in a similar study involving cellsurface proteomics [22], proteins that were annotated as“plasma membrane,” “cell surface,” and “extracellular”in the database were all considered cell surface proteins.Then, the selected proteins were filtered by N-linkedglycosylation site prediction (NXS/T sequence motifs)and O-linked glycosylation site prediction using CBSPrediction servers (http://www.cbs.dtu.dk/services/).

Real-time PCR and Western blot analysis

The messenger RNA of different cell lines was isolatedusing a PolyATtract System 1000 (Promega, USA) andreversely transcribed to complementary DNA using a Pri-meScript RT reagent kit (TaKaRa, Japan). Real-time PCRwas performed using an SYBR Premix EX Taq II kit(TaKaRa, Japan) in a final volume of 25 μL. The data wereacquired and analyzed using a Mastercycler ep realplexsystem (Eppendorf, Germany). All the primers used in this

Cell surface glycoprotein profiling of cancer cells 1663

research are listed in the electronic supplementary materialand were applied with the proper program.

Whole-cell lysates were prepared using radioimmunopre-cipitation assay lysis buffer, quantified, and diluted to 1 mg/mL. After electrophoretic separation and transfer to poly(vinylidene difluoride) membranes, the proteins weredetected with a primary antibody and then with a secondaryantibody (the antibodies were obtained from Abcam, andCell Signaling, and are listed in Table S1).

Immunofluorescence assays

The cells were washed three times with PBS, fixed with 4 %paraformaldehyde/PBS for 15 min, and then blocked with3 % skim milk/PBS for 30 min. The cells were incubatedovernight with an anti-NrCAM antibody (ab24344, Abcam,UK, 1:1,000 diluted) and visualized using a fluoresceinisothiocyanate conjugated anti-rabbit antibody (sc-2012,Santa Cruz, USA).

Immunohistochemistry

Immunohistochemistry was applied to investigate NrCAMexpression in NSCLC, cervical carcinoma, and pancreaticcarcinoma tissues. Paraffin blocks of tissue chips, whichincluded tumor tissues and the same amounts of homolo-gous adjacent noncancerous tissues, were obtained fromShanghai Outdo Biotech and were detected with NrCAMantibody (Santa Cruz, USA) diluted 1:100. The proportionsof immunoreactive cells were categorized as less than 5 %,5–25 %, 26–50 %, 51–75 %, 76–95 %, or more than 95 %reactive cells. Statistical analyses of the relationships be-tween NrCAM expression and clinical features were per-formed using the χ2 test.

Results

Fluorescent labeling of cell surface glycoproteins

Two alkyne probes, biotin-LC-alkyne and biotin-SS-alkyne,were synthesized for the biotinylation of cell surface glyco-proteins in cells that metabolized with azides. The specific-ity of this reaction and the release of biotin-SS-alkyne byreductive reaction were determined by a fluorescentvisualization.

As shown in Fig. 2, after reaction with probes and fluo-rescent labeling, cells that metabolized Ac4ManNAz clearlydisplayed cell surface fluorescence (Fig. 2b, e), whereas avery low fluorescence intensity was observed in the controlgroup, which was cultured with Ac4ManNAc (Fig. 2a, d).This result suggested that the increased fluorescence wasdue to a specific affinity for azido glycans and that the azido

groups present on cell surfaces might undergo a highlyselective reaction with both alkyne probes. In the DTT-treated group, the fluorescence was lost in cells that reactedwith biotin-SS-alkyne (Fig. 2c), whereas cells that reactedwith biotin-LC-alkyne maintained their fluorescence inten-sities (Fig. 2f). This result suggested that the probe contain-ing the disulfide bond was completely released by anappropriate reductive reaction.

Figure 2g shows the clear fluorescent region of cells thatreacted with biotin-SS-alkyne. Although the cells were per-meabilized, no fluorescence was observed in the cytoplasm.The biotin-LC-alkyne-labeled cells displayed the sameresults (data not shown), which indicated that both probesdid not penetrate the cell membrane and that only themolecules containing azido sugars were labeled (avidin–Cy3 was demonstrated to have the ability to penetrate per-meabilized cells, as described in the electronic supplemen-tary material). All the above results indicated that the azido–alkyne–biotin system was specific, and that it is feasible todevelop a procedure based on this system for glycoproteinpurification.

Isolation of glycoproteins

Biotin-SS-alkyne was applied in the procedure to purify cellsurface glycoproteins because proteins biotinylated withbiotin-SS-alkyne could be conveniently released, and thisdetachment from the resin was helpful to identify moreproteins [23] (Fig. 3a). Sodium dodecyl sulfate polyacryl-amide gel electrophoresis was performed to detect glyco-proteins enriched from HeLa cells (Fig. 3b). The resultsshowed that abundant proteins were enriched from Ac4Man-NAz-metabolized cells (line 2); however, only a few bandscould be detected in the control sample, which wascultured with Ac4ManNAc (line 1). This result indicatedthat azido-metabolized proteins were selectively enriched bythis purification procedure. Generally, approximately50 μg of glycoprotein could be collected with 1 mLof beads.

Identification and analysis of glycoproteins

To identify cancer-related glycoproteins, glycoproteins wereisolated from three different human cancer cell lines, and theproteins were then analyzed by high-performance liquidchromatography–MS/MS. In this analysis, 310 proteinswere identified (155 proteins of A549 cells; 187 proteinsfor HeLa cells; 204 proteins for SW1990 cells; uniquepeptides count, 2 or greater).

As described above, localization and glycosylation werepredicted using bioinformatics tools. Fifty-six proteins werepredicted to be present on the cell surface and to contain N-glycosylation or O-glycosylation sites (Table 1). Although

1664 P. Pan et al.

these proteins were predicted to contain one or more glyco-sylation sites, these sites might be glycosylated at differentlevels even without glycosylation in diverse cell lines. Re-gardless, this result provided a glycoprotein expression pat-tern for tumor cell lines, which was helpful for investigatingthe expression and glycosylation of these proteins in cancerdevelopment.

NrCAM expression in A549 cells

From the liquid chromatography–MS/MS results, NrCAMpeptides were only detected in the A549 product (see theelectronic supplementary material). Real-time PCR and

Western blotting were performed to detect the expressionlevels of proteins in the three cell lines. As with the MS data,NrCAM was not detected in HeLa or SW1990 cells(Fig. 4a). As the strategy focused on cell surface proteins,we verified NrCAM expression on the A549 cell surface byimmunofluorescence. A549 cells were fixed and detectedwith an NrCAM antibody. Because HSP47 is predominantlyexpressed in the endoplasmic reticulum and is absent at thecell surface, we used the HSP47 antibody as a control. Wefound that NrCAM was located in restricted areas on the cellsurface of A549 cells, and that NrCAM might be anchoredin some type of structure such as a lipid raft in the plasmamembrane (Fig. 4b).

Fig. 2 HeLa cells were grown with 200 mM peracetylated N-acetyl-mannosamine (Ac4ManNAc) (a, d) or 200 mM peracetylated N-azi-doacetylmannosamine (Ac4ManNAz) (b, c, e, f). The cells in a–c werereacted with biotin-SS-alkyne and the cells in d–f were reacted withbiotin-LC-alkyne. Then the biotinylated molecules were detected with

avidin–Cy3. The cells in c and f underwent an additional dithiothreitol(DTT) treatment after fluorescent labeling. g The fluorescent locationin a biotin-SS-alkyne-reacted HeLa cell. The cell nucleus was stainedwith 4′,6-diamidino-2-phenylindole (DAPI)

Fig. 3 Strategy for the affinity enrichment of cell surface glycopro-teins (a). Cells were metabolized with Ac4ManNAz and biotinylatedwith an alkyne probe. After affinity enrichment using streptavidinbeads, the glycoproteins were released with DTT. This strategy wasapplied to HeLa cells. Glycoproteins purified from 2 mg lysate of cells

metabolized with Ac4ManNAz (b, lane 2), or without as a control (b,lane 1), were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis. Two micrograms of lysate (b, lane 3, cultured withAc4ManNAc; b, lane 4, cultured with Ac4ManNAc) was also loaded todemonstrate the relative enrichment

Cell surface glycoprotein profiling of cancer cells 1665

Table 1 The proteins identified were grouped by their location on the membrane and predictions of N-glycosylation and O-glycosylation sites

Access no. Gene symbol and gene name Cell lines Glycosylation site

HeLa A549 SW1990

P31947 SFN, 14-3-3 protein sigma MS — MS N

P12814 ACTN1, α-actinin 1 — MS MS N, O

O43707 ACTN4, α-actinin 4 MS MS MS N

P04075 ALDOA, fructose bisphosphate aldolase A — MS MS O

P09923 ALPI, intestinal-type alkaline phosphatase MS — — N

P05186 ALPL, alkaline phosphatase, tissue-nonspecific isozyme MS — — N

P04083 ANXA1, annexin A1 MS MS MS N, O

P07355 ANXA2, annexin A2 MS MS MS N

P84077 ARF1, ADP-ribosylation factor 1 MS MS MS N

P25705 ATP5A1, ATP synthase subunit α MS MS MS N

P06576 ATP5B, ATP synthase subunit β MS MS MS O

P80723 BASP1, brain acid soluble protein 1 MS — — O

P27797 CALR, calreticulin MS — — N

O43852 CALU, calumenin MS — — N

Q01518 CAP1, adenylyl cyclase associated protein 1 — — MS N, O

Q6YHK3 CD109 antigen MS MS — N

P16070 CD44 antigen MS — — N, O

Q00610 CLTC, clathrin heavy chain 1 — MS MS N, O

P10909 CLU, clusterin — MS — N

Q12860 CNTN1, contactin 1 — MS — N, O

P06733 ENO1, α-enolase MS MS MS N

P21333 FLNA, filamin A MS MS MS N, O

Q13283 G3BP1, Ras GTPase-activating protein-binding protein 1 — — MS N, O

P16190 HLA class I histocompatibility antigen, A-33 α chain MS — — N, O

P30462 HLA class I histocompatibility antigen, B-14 α chain MS — — N, O

P01112 HRAS, GTPase HRas MS — — N

P14625 HSP90B1, endoplasmin MS MS MS N

P11142 HSPA8, heat shock cognate 71-kDa protein MS MS MS N

P38646 HSPA9, stress-70 protein, mitochondrial MS MS MS N, O

P04792 HSPB1, heat shock protein β1 MS MS MS O

P10809 HSPD1, 60-kDa heat shock protein, mitochondrial MS MS MS N, O

Q92598 HSPH1, heat shock protein 105 kDa MS — MS N, O

P46940 IQGAP1, Ras GTPase-activating-like protein IQGAP1 MS MS MS N, O

P09382 LGALS1, galectin 1 MS — — N

P17931 LGALS3, galectin 3 MS — MS N, O

P29966 MARCKS, myristoylated alanine-rich C-kinase substrate MS — — N, O

O00159 MYO1C, myosin Ic — MS — N, O

Q92823 NRCAM, neuronal cell adhesion molecule — MS — N, O

P07237 P4HB, protein disulfide isomerase MS — MS O

Q9UKS6 PACSIN3, protein kinase C and casein kinase substrate in neurons protein 3 — MS — O

P35232 PHB, prohibitin MS MS MS N

P62937 PPIA, peptidyl-prolyl cis–trans isomerase A MS MS MS N

P61026 RAB10, Ras-related protein Rab-10 — MS — N

P51148 RAB5C, Ras-related protein Rab-5C — MS — N

P13489 RNH1, ribonuclease inhibitor MS MS — N

P36952 SERPINB5, serpin B5 — — MS N

P11166 SLC2A1, solute carrier family 2, facilitated glucose transporter member 1 — — MS N, O

1666 P. Pan et al.

NrCAM immunohistochemistry

We further investigated the expression of NrCAM inNSCLC, cervical carcinoma, and pancreatic carcinoma tis-sues. The proportions of reactive cells were counted, and theresults are listed in Table 2. NrCAM was found to bemoderately expressed in both NSCLC samples and adjacentnormal tissues; however, a negative result was obtainedfrom cervical carcinoma, pancreatic carcinoma, and thecorresponding noncancerous tissues (Fig. 5). This resultcoincided with the MS data, indicating that NrCAM wasonly detected in NSCLC cell line A549. In NSCLC cases,no significant diversity was found in noncancerous tissuesadjacent to adenocarcinomas and squamous cells [χ20

5.895, four degrees of freedom (df), P00.207]. The positiveratio of NrCAM displayed a significant increase in adeno-carcinoma tissues (χ2028.515, df04, P<0.01) comparedwith adjacent noncancerous tissues. However, this alteration

was not detected in squamous carcinomas (χ207.579, df04,P00.108). This result indicated that a wide expression ofNrCAM might be a specific feature of adenocarcinomas inNSCLC.

Discussion

Previous studies based on bioorthogonal chemistry appliedchemical modification of various monosaccharaides, such asmannose, fucose, galactose, and glucose [24]. With use ofthis technique, glycoproteins metabolized with certain sub-units were visualized and trafficked in vitro and in vivo [25]and captured in glycoproteomic studies [26]. In this study,we utilized Ac4ManNAz for the purification of cell surfaceglycoproteins and aimed to identify specific biomarkers indifferent cancer cell lines.

Table 1 (continued)

Access no. Gene symbol and gene name Cell lines Glycosylation site

HeLa A549 SW1990

P08195 SLC3A2, 4F2 cell-surface antigen heavy chain MS MS MS N

Q01650 SLC7A5, large neutral amino acids transporter small subunit 1 MS MS — N

O14745 SLC9A3R1, Na+/H+ exchange regulatory cofactor NHE-RF1 MS MS — O

Q01082 SPTBN1, spectrin β chain, brain 1 — MS MS N, O

P02786 TFRC, transferrin receptor protein 1 MS — MS N, O

Q9Y490 TLN1, talin 1 MS MS — N, O

P54578 USP14, ubiquitin carboxyl-terminal hydrolase 14 — — MS N

P18206 VCL, vinculin MS — — N, O

P54577 YARS, tyrosyl-transfer RNA synthetase — — MS N, O

MS mass spectrometry

Fig. 4 Western blotting and real-time PCR were applied to identifyexpression levels in the three cancer cell lines. The real-time PCRanalysis (upper histogram) was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a). Cell surface expression wasdetected using a fluorescein isothiocyanate (FITC)-labeled anti-

neuronal cell adhesion molecule (NrCAM) IgG that recognizes anextracellular region of NrCAM (b), and an FITC-labeled antibodyagainst HSP47, which is expressed in the endoplasmic reticulum,was employed as a negative control (c). The cell nucleus was stainedwith DAPI

Cell surface glycoprotein profiling of cancer cells 1667

Sulfo-NHS-LC-biotin and sulfo-NHS-SS-biotin havebeen used in proteomic studies involving the cell surface,but these methods were unable to distinguish nonglycopro-teins from glycoproteins. In this study, we expected todevelop a method for separation of glycoproteins. Scheureret al. [23] have reported a comparison of strategies employ-ing these two reagents and demonstrated that digestingsulfo-NHS-SS-biotin-labeled proteins eluted from a resin

results in more identified proteins than on-resin digestionno matter whether the proteins were labeled with sulfo-NHS-LC-biotin or sulfo-NHS-SS-biotin. That study wasinstructive for the purification procedure used in this studybecause biotinylated reagents with a similar structure wereused here. In the fluorescent detection, both biotin-LC-alkyne and biotin-SS-alkyne displayed high specificitiesfor azido-labeled cells and were restricted to the cell surface.

Table 2 Proportion of neuronal cell adhesion molecule (NrCAM)expression in tumor and noncancerous tissues of cancer types involvedin this study. NrCAM was widely expressed in non-small-cell lung(NSCL) adenocarcinomas compared with their adjacent noncancerous

tissue (P<0.05). However, such an alternation was found in NSCLsquamous carcinomas. No NrCAM expression was found in cervicalcarcinoma and pancreatic carcinoma

<5 % 5-25 % 26-50 % 51-75 % >75 %

NSCL adenocarcinomas (n029) 1 3 4 4 17

Adjacent noncancerous tissue (n029) 4 16 7 1 1

NSCL squamous carcinoma (n030) 1 13 6 6 4

Adjacent noncancerous tissue (n030) 4 13 13 3 0

Cervical carcinoma (n031) 31 0 0 0 0

adjacent noncancerous tissue (n031) 31 0 0 0 0

Pancreatic carcinoma (n031) 30 1 0 0 0

Adjacent noncancerous tissue (n031) 30 1 0 0 0

Fig. 5 Representativeimmunostaining resultsobtained using anti-NrCAMantibodies (clone sc-18958,Santa Cruz) on sections oftumorous and adjacent noncan-cerous tissues: a non-small-celllung squamous carcinoma;b non-small-cell lung adeno-carcinomas; c pancreatic carci-noma; d cervical carcinoma.NrCAM is mainly localized tothe plasma membrane andcytoplasm. Its expression wasincreased in adenocarcinomasand compared with adjacentnoncancerous tissue. Most ofthe non-small-cell lung carci-nomas and their correspondingnormal lung tissues wereweakly stained

1668 P. Pan et al.

Biotin-SS-alkyne was then chosen for the following captureof glycoproteins to gain further information.

After high-performance liquid chromatography–MS/MS,56 of 310 identified proteins were predicted to be located onthe cell surface and to contain at least one glycosylation site.Although a number of glycoproteins were identified, therewere many proteins that were neither glycosylated nor locatedon the cell surface. In previous studies using biotinylationreagents, researchers suggested that contamination eventsmight be induced by the uptake of the biotin agent or byleakage from damaged cells, which induce faulty reactions.A staining procedure with trypan blue was performed duringthe Cu(I)-catalyzed [3+2] cycloaddition reaction. Almost allthe cells remained intact even when the reaction was extendedto 90 min (data not shown). Because only nonviable cellscould be stained, the reaction procedure appeared to notdamage cell integrity during the experiment. Fluorescentimaging also indicated a highly specific biotin activity onthe cell surface, which could be metabolized with the azidogroup. Furthermore, nonglycosylated proteins could not bebiotinylated using this strategy. Therefore, a faulty reactionmight not be the primary cause of the contamination. In thisstudy, the beads were washed sufficiently with 1 M KClduring the isolation procedure, but weak bands were stilldetected in the control sample. This phenomenon wasdetected even though magnetic beads and avidin–agarosefrom different companies were utilized, which suggested anonspecific binding potential for the beads or an endogenousbiotin activity, both of which might interfere with the capture.Moreover, with the purpose to identify more glycoproteins,the lysate was prepared with no detergent and was centrifugedat a low speed after sonication. However, this procedure mightinduce most of the insoluble content or cellular complexes inthe lysate and finally result in linking of such forms to thebeads. Although a significant proportion of nonglycosylatedproteins was detected, this study provided us with a group ofcell surface glycoproteins that were expressed in a particularpattern in different cell lines.

The glycoproteins identified are involved in cell adhe-sion, transport, and other biological process. Nearly half ofthe glycoproteins coexist in two or three cell lines, butothers were found to be uniquely expressed in a certain cellline. In our results, CNTN1 and NrCAM were only detectedin the A549 non-small-cell lung adenocarcinoma cell line.CNTN1 and NrCAM belong to the immunoglobulin-likecell adhesion molecule family, and both are predominantlyexpressed in the central nervous system [27]. Heterophilicbinding between NrCAM and CNTN1 is found in nervoustissues, and this complex is reported to enhance neuriteoutgrowth and sensory axon guidance [28]. Recently,CNTN1 and NrCAM were also observed to be overex-pressed in certain carcinomas and involved in tumor inva-sion and metastasis. In lung adenocarcinomas, CNTN1

expression is positively associated with cell invasiveness,and knocking down CNTN1 expression suppressed tumorinvasiveness and metastasis [29]. NrCAM has been detectedin pancreatic cancer, melanoma, renal carcinoma, and coloncarcinoma [30], and its expression has also been found toenhance the motility and proliferation of tumor cells [31].However, there has never been a report describing the ex-pression of NrCAM in NSCLC. In our study, we observedNrCAM upregulation in lung adenocarcinoma, which sug-gests that CNTN1 and NrCAM might function in lungadenocarcinoma as a heterodimeric complex.

As reported, NrCAM induces neurite outgrowth throughreceptor-type protein tyrosine phosphatase β (RPTPβ),which belongs to receptor-type protein tyrosine phospha-tases, by forming a complex with CNTN1 [28]. RPTPβ is agrowth factor that is also primarily detected in the nervoussystem and is known to be involved in controlling cellproliferation, migration, and apoptosis [32]. Recently, Fenget al. [33] reported that RPTPβ is also involved in lungcancer cell migration. In A549 cells, transfection with meninrepresses RPTPβ, thereby inhibiting cell migration. Whenall these factors are considered, one possibility is thatNrCAM and CNTN1 might function by activating RPTPβin lung adenocarcinoma, which has been observed in thenervous system but rarely investigated in tumor cells. Fur-ther investigations will help us to better understand howthese proteins work to regulate tumor behavior.

Acknowledgments This work was supported in part by the NationalKey Basic Research and Development Program of China (973 Pro-gram) (2007CB914803) and the National Natural Science Foundationof China (30900256, 31170768).

References

1. Apweiler R, Hermjakob H, Sharon N (1999) Biochim BiophysActa Gen Subj 1473:4–8

2. Varki A (1993) Glycobiology 3:97–1303. Dube DH, Bertozzi CR (2005) Nat Rev Drug Discov 4:477–4884. Whelan SA, Lu M, He JB, Yan WH, Saxton RE, Faull KF, White-

legge JP, Chang HR (2009) J Proteome Res 8:4151–41605. Fang XM, Zhang WW (2008) J Proteomics 71:284–3036. Plavina T, Wakshull E, Hancock WS, Hincapie M (2007) J Pro-

teome Res 6:662–6717. Zhang Q, Tang N, Brock JW, Mottaz HM, Ames JM, Baynes JW,

Smith RD, Metz TO (2007) J Proteome Res 6:2323–23308. Wells L, Vosseller K, Cole RN, Cronshaw JM, Matunis MJ, Hart

GW (2002) Mol Cell Proteomics 1:791–8049. Palmisano G, Lendal SE, Larsen MR (2011) Methods Mol Biol

753:309–32210. Pompach P, Chandler KB, Lan R, Edwards N, Goldman R (2012) J

Proteome Res 11:1728–174011. Prescher JA, Bertozzi CR (2005) Nat Chem Biol 1:13–2112. Prescher JA, Bertozzi CR (2006) Cell 126:851–85413. Saxon E, Bertozzi CR (2000) Science 287:2007–201014. Sarkar AK, Fritz TA, Taylor WH, Esko JD (1995) Proc Natl Acad

Sci USA 92:3323–3327

Cell surface glycoprotein profiling of cancer cells 1669

15. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002)Angew Chem Int Ed 41:2596–2599

16. Chang PV, Chen X, Smyrniotis C, Xenakis A, Hu T, Bertozzi CR,Wu P (2009) Angew Chem Int Ed 48:4030–4033

17. Luchansky SJ, Argade S, Hayes BK, Bertozzi CR (2004) Bio-chemistry 43:12358–12366

18. Chang PV, Prescher JA, Sletten EM, Baskin JM, Miller IA,Agard NJ, Lo A, Bertozzi CR (2010) Proc Natl Acad SciUSA 107:1821–1826

19. Sampathkumar SG, Li AV, Yarema KJ (2006) Nat Protoc1:2377–2385

20. Dai J, Shieh CH, Sheng QH, Zhou H, Zeng R (2005) Anal Chem77:5793–5799

21. Bhatia VN, Perlman DH, Costello CE, McComb ME (2009) AnalChem 81:9819–9823

22. Gu B, Zhang J, Wang W, Mo L, Zhou Y, Chen L, Liu Y, Zhang M(2010) PLoS One 5:e15795

23. Scheurer SB, Roesli C, Neri D, Elia G (2005) Proteomics5:3035–3039

24. Sawa M, Hsu TL, Itoh T, Sugiyama M, Hanson SR, Vogt PK,Wong CH (2006) Proc Natl Acad Sci USA 103:12371–12376

25. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR (2008) Sci-ence 320:664–667

26. Hanson SR, Hsu TL, Weerapana E, Kishikawa K, Simon GM,Cravatt BF, Wong CH (2007) J Am Chem Soc 129:7266–7267

27. Grumet M (1997) Cell Tissue Res 290:423–42828. Sakurai T, Lustig M, Nativ M, Hemperly JJ, Schlessinger J, Peles

E, Grumet M (1997) J Cell Biol 13:907–91829. Su JL, Yang CY, Shih JY, Wei LH, Hsieh CY, Jeng YM, Wang MY,

Yang PC, Kuo ML (2006) Cancer Res 66:2553–256130. Conacci-Sorrell M, Zhurinsky J, Ben-Ze'ev A (2002) J Clin Invest

109:987–99131. Conacci-Sorrell ME, Ben-Yedidia T, Shtutman M, Feinstein E,

Einat P, Ben-Ze'ev A (2002) Genes Dev 16:2058–207232. Perez-Pinera P, Chang Y, Deuel TF (2007) Cell Cycle

6:2877–288333. Feng ZJ, Gao SB, Wu Y, Xu XF, Hua X, Jin GH (2010) Oncogene

29:5416–5426

1670 P. Pan et al.