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Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

2008; Elsevier BV; Volume: 8; Issue: 1 Linguagem: Inglês

10.1074/mcp.m800308-mcp200

ISSN

1535-9484

Autores

Atsushi Kuno, Yukinari Kato, Atsushi Matsuda, Mika K. Kaneko, Hiromi Ito, Koh Amano, Yasunori Chiba, Hisashi Narimatsu, Jun Hirabayashi,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery. Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery. Glycan synthesis in individual cells is regulated by harmonized expression of more than a hundred glycosyltransferases. Importantly, detectable dynamics occurring on each cell surface during diverse biological events, e.g. differentiation, proliferation, and signal induction, indicate drastic changes in the glyco-machinery (1Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar). During the last few decades, there have been enormous advances in the findings of glycosylation alterations related to oncogenesis. Cell surface sialylation and β1–6 branching of N-linked glycans are strongly correlated with metastatic potential of cancer cells (2Dennis J. Waller C. Timpl R. Schirrmacher V. 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Proteomics. 2006; 5: 1957-1967Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). This has resulted in the emergence of an extensive range of biomarker “candidates”, many of which are glycoproteins. However, these candidate molecules need to be subsequently subjected to a verification step prior to a much large scale validation phase (e.g. treating with >1,000 incidences). Antibody microarray has taken the place of ELISA as a more versatile and high-throughput technique, and has enabled multiplexed quantitative analysis for over a hundred proteins with sufficient sensitivity (15Haab B.B. Antibody arrays in cancer research.Mol. Cell. Proteomics. 2005; 4: 377-383Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). As an application of MS technology, multiple-reaction monitoring-MS has also been developed (16Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. 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Proteomics. 2008; 7: 1974-1982Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). However, taking into consideration the critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al. (20Chen S. LaRoche T. Hamelinck D. Bergsma D. Brenner D. Brand R.E. Haab B.B. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays.Nat. Methods. 2007; 4: 437-444Crossref PubMed Scopus (86) Google Scholar) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated assays with more than 30 lectins to search for critical differences in glycan structures. In the present study, an alternative approach, i.e. “antibody-overlay lectin microarray” (21Rosenfeld R. Bangio H. Gerwig G.J. Rosenberg R. Aloni R. Cohen Y. Amor Y. Plaschkes I. Kamerling J.P. Maya R.B. A lectin array-based methodology for the analysis of protein glycosylation.J. Biochem. Biophys. Methods. 2007; 70: 415-426Crossref PubMed Scopus (95) Google Scholar), was taken, which is based on the lectin microarray platform originally developed in our laboratory (22Kuno A. Uchiyama N. Koseki-Kuno S. Ebe Y. Takashima S. Yamada M. Hirabayashi J. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.Nat. Methods. 2005; 2: 851-856Crossref PubMed Scopus (439) Google Scholar, 23Uchiyama N. Kuno A. Koseki-Kuno S. Ebe Y. Horio K. Yamada M. Hirabayashi J. Development of a lectin microarray based on an evanescent-field fluorescence principle.Methods Enzymol. 2006; 415: 341-351Crossref PubMed Scopus (37) Google Scholar, 24Uchiyama N. Kuno A. Tateno H. Kubo Y. Mizuno M. Noguchi M. Hirabayashi J. Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins.Proteomics. 2008; 8: 3042-3050Crossref PubMed Scopus (51) Google Scholar). In this strategy, a specific glycan profile of a target glycoprotein is acquired with the aid of a specific antibody raised against the core protein moiety, removing the need to covalently label each glycoprotein with a fluorescent reagent (Refs. 22Kuno A. Uchiyama N. Koseki-Kuno S. Ebe Y. Takashima S. Yamada M. Hirabayashi J. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.Nat. Methods. 2005; 2: 851-856Crossref PubMed Scopus (439) Google Scholar, 23Uchiyama N. Kuno A. Koseki-Kuno S. Ebe Y. Horio K. Yamada M. Hirabayashi J. Development of a lectin microarray based on an evanescent-field fluorescence principle.Methods Enzymol. 2006; 415: 341-351Crossref PubMed Scopus (37) Google Scholar, 24Uchiyama N. Kuno A. Tateno H. Kubo Y. Mizuno M. Noguchi M. Hirabayashi J. Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins.Proteomics. 2008; 8: 3042-3050Crossref PubMed Scopus (51) Google Scholar, 25Angeloni S. Ridet J.L. Kusy N. Gao H. Crevoisier F. Guinchard S. Kochhar S. Sigrist H. Sprenger N. Glycoprofiling with microarrays of glycoconjugates and lectins.Glycobiology. 2005; 15: 31-41Crossref PubMed Scopus (315) Google Scholar, 26Pilobello K.T. Krishnamoorthy L. Slawek D. Mahal L.K. Development of a lectin microarray for the rapid analysis of protein glycopatterns.ChemBioChem. 2005; 6: 985-989Crossref PubMed Scopus (248) Google Scholar, 27Ebe Y. Kuno A. Uchiyama N. Koseki-Kuno S. Yamada M. Sato T. Narimatsu H. Hirabayashi J. Application of lectin microarray to crude samples: differential glycan profiling of Lec mutants.J. Biochem. 2006; 139: 323-327Crossref PubMed Scopus (58) Google Scholar, 28Hsu K.L. Pilobello K.T. Mahal L.K. Analyzing the dynamic bacterial glycome with a lectin microarray approach.Nat. Chem. Biol. 2006; 2: 153-157Crossref PubMed Scopus (212) Google Scholar, 29Pilobello K.T. Slawek D.E. Mahal L.K. A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 11534-11539Crossref PubMed Scopus (177) Google Scholar and Fig. 1A). Once a target glycoprotein (<100 ng) is enriched from a trace amount of crude samples by a microliter-scale immunoprecipitation procedure with the biotinylated antibody used for the above detection, a precise glycan profile of the target protein is obtained in a highly reproducible and high-throughput manner (see Fig. 1B). The system is unique in its ability to distinguish critical differences in both core and terminal structures occurring on N- and O-glycans attached to the proteins. The combined procedure, consisting of antibody-enrichment, quantification and overlay detection of lectin microarray, is designated antibody-assisted lectin profiling (ALP). The system provides a feasible and powerful advance toward “focused differential glycoproteomics”. Reagents were purchased from Sigma, Wako Chemicals (Osaka, Japan), or Nacalai Tesque (Kyoto, Japan) unless otherwise noted. Seminal plasma PSA and human serum transferrin (hTf) were obtained from R&D Systems, Inc. (Minneapolis, MN) and Athens Research & Technology (Athens, GA), respectively. Biotinylated mouse anti-PSA monoclonal antibody (bio-αPSA) and rabbit anti-hTf polyclonal antibody (bio-αhTf) were obtained from R&D Systems, Inc. and Abcam, Inc. (Cambridge, MA), respectively. Chinese hamster ovary (CHO) and prostate cancer cell lines (LNCaP and PCa2b) were obtained from the American Type Culture Collection (ATCC). Fifteen glioblastoma cell lines (LN18, LN215, LN229, LNZ308, LN319, LN340, LN428, LN464, U87, U178, U251, U373, A1207, SF763, and T98G) were donated by Dr. Webster K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA). Two prostate cancer cell lines (PC3, DU145) were obtained from Tohoku University. Lymphatic endothelial cells (LECs) were purchased from AnioBio (Del Mar, CA). FLAG-tagged hPod-transfected CHO cell line was prepared as described previously (30Kaneko M.K. Kato Y. Kameyama A. Ito H. Kuno A. Hirabayashi J. Kubota T. Amano K. Chiba Y. Hasegawa Y. Sasagawa I. Mishima K. Narimatsu H. Functional glycosylation of human podoplanin: glycan structure of platelet aggregation-inducing factor.FEBS Lett. 2007; 581: 331-336Crossref PubMed Scopus (90) Google Scholar). These cell lines were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium (for CHO, PC3, and DU145) or Dulbecco’s modified Eagle’s medium (for glioblastoma cell lines, LNCaP and PCa2b) supplemented with 1 or 10% heat-inactivated fetal bovine serum (Sigma), 2 mm l-glutamine (Invitrogen), and antibiotics (100 μg/ml of kanamycin for CHO, 100 IU/ml of penicillin, and 100 μg/ml of streptomycin for PC3, DU145, LNCaP, and PCa2b). For a pilot experiment of lectin microarray analysis using tissue sections, glioblastoma cell line LN319 was transplanted into BALB/c nude mice. The grown glioblastoma-like tumor was dissected at 5 μm for immunohistochemical and lectin microarray analyses. Seminoma specimen was purchased from Cybrdi, Inc. (Frederick, MD). hPod/CHO and hPod/LN319 were purified as described by Kato et al. (31Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain.Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (171) Google Scholar). Briefly, cultured cells (1 × 1010 cells/preparation) were lysed with PBS containing 0.25% Triton X-100. The lysate from CHO was applied on anti-FLAG antibody-conjugated-agarose gel (M2; Sigma). After washing the gel, the captured hPod/CHO was eluted with 100 μg/ml FLAG peptide (Sigma). To purify hPod/LN319, the cell lysate was applied on NZ-1 antibody-immobilized resin. After washing the gel, the captured hPod/CHO was eluted with hpp38–51 peptide (EGGVAMPGAEDDVV) (31Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain.Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (171) Google Scholar). A set of recombinant hPods with defined O-glycan structures were prepared as described by Amano et al. (32Amano K. Chiba Y. Kasahara Y. Kato Y. Kaneko M.K. Kuno A. Ito H. Kobayashi K. Hirabayashi J. Jigami Y. Narimatsu H. Engineering of mucin-type human glycoproteins in yeast cells.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3232-3237Crossref PubMed Scopus (71) Google Scholar). Briefly, recombinant glutathione S-transferase and His6-tagged hPods with Tn and Core1 structures were expressed in TN-1 and T-1 cells, respectively. The hPods purified by affinity chromatography were digested with Precision protease. After removing glutathione S-transferase tag fragments, both recombinant hPods were subsequently reacted with appropriate glycosyltransferases to build up STn and Core3 structures on the hPod having Tn and Core2, sialylated T (ST) and disialylated T antigen (diST) structures on the hPod having T (Core1), respectively. Tissue sections on glass slides were deparaffinized and rehydrated. Testicular tumor specimens were incubated first with NZ-1 for Pod (a rat monoclonal antibody; 1 μg/ml) at room temperature for 1 h, then with biotin-conjugated secondary anti-rat IgG antibody (Dako, Glostrup, Denmark) for 1 h, and finally with peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA) for 1 h. Color was developed using 3,3′-diaminobenzene tetrahydrochloride tablet sets (Dako) for 3 min. In the case of PSA, prostate cancer or benign prostate hyperplasia specimens were incubated with bio-αPSA at room temperature for 1 h. For analysis of endogenous hPod, each cell line was solubilized with lysis buffer (1% Triton X-100 in PBS (PBSTx)). Immunohistochemically hPod-positive cell populations from tissue sections were excised (about 3.5 mm3) and solubilized with ProteoSOL Tissue Extraction System (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The hPods in these lysates were immunoprecipitated using 10 μg of NZ-1 antibody immobilized-Sepharose. After washing the resin with 1 ml of PBS, captured hPods were released with 20 μl of SDS-PAGE sample buffer excluding bromphenol blue, reducing agent and glycerol by heat denaturation. For PSA analysis, PSA was immunoprecipitated from culture supernatant concentrate (20 times) of each cell line using 500 ng of biotinylated anti-PSA monoclonal antibody pre-conjugated to 10 μl of streptavidin-immobilized magnetic beads, Dynabeads MyOne™ Streptavidin T1 (DYNAL Biotech ASA, Oslo, Norway) conjugate. The beads thus capturing PSA were washed three times with 200 μl of PBSTx, and then the bound PSA was eluted with 10 μl of the elution buffer (TBS containing 0.2% SDS) by heat denaturation. After dilution by an equal volume of 1% Triton X-100 in TBS, the contaminant antibody was completely depleted by addition of 20 μl of the streptavidin-immobilized magnetic beads. The purified hPods were electrophoresed under reducing conditions on 10–20% polyacrylamide gels. The separated proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 4% skim milk in PBS, the membrane was incubated with NZ-1 antibody, then with peroxidase-conjugated anti-rat antibody (1/1000 diluted; GE Healthcare) and developed for 1 min with ECL reagents (GE Healthcare) using Kodak X-Omat AR film. In the case of PSA, the membrane was incubated with biotinylated anti-PSA monoclonal antibody, then with alkaline phosphatase-conjugated streptavidin (1/5000 diluted with TBST; ProZyme, Inc., San Leandoro, CA). The treated membrane was reacted with Western Blue® stabilized substrate for alkaline phosphatase (Promega). Antibody-overlay lectin microarray was basically performed as described previously (22Kuno A. Uchiyama N. Koseki-Kuno S. Ebe Y. Takashima S. Yamada M. Hirabayashi J. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.Nat. Methods. 2005; 2: 851-856Crossref PubMed Scopus (439) Google Scholar, 23Uchiyama N. Kuno A. Koseki-Kuno S. Ebe Y. Horio K. Yamada M. Hirabayashi J. Development of a lectin microarray based on an evanescent-field fluorescence principle.Methods Enzymol. 2006; 415: 341-351Crossref PubMed Scopus (37) Google Scholar, 24Uchiyama N. Kuno A. Tateno H. Kubo Y. Mizuno M. Noguchi M. Hirabayashi J. Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins.Proteomics. 2008; 8: 3042-3050Crossref PubMed Scopus (51) Google Scholar). Briefly, the purified proteins were diluted to 60 μl with PBSTx or TBSTx and then applied to the lectin array containing triplicate spots of 43 lectin (see supplemental Fig. S1) into each of 8-divided incubation baths on the epoxy-coated glass slide from Schott AG (Mainz, Germany). After incubation at 20 °C for 12 h, 20 μg of human serum polyclonal IgG was added to the glass slide followed by 30-min incubation. The reaction solution was discarded, and the glass slide was washed three times with PBSTx. 60 μl of biotinylated antibody solution in PBSTx was applied to the array and then incubated at 20 °C for 1 h. After washing three times with PBSTx, 60 μl of Cy3-labeled streptavidin (GE Healthcare) solution in PBSTx was added to the array and then incubated at 20 °C for 30 min. The glass slide was rinsed with PBSTx and scanned by an evanescent-field fluorescence scanner, GlycoStation™ (Moritex, Co., Tokyo, Japan). All data were analyzed with the Array-Pro analyzer version 4.5 (Media Cybernetics, Inc.). The net intensity value for each spot was calculated by subtracting background value from signal intensity values of three spots. To remove Sia residues, hPods were incubated with Sialidase A™ from Arthrobacter ureafaciens (ProZyme, Inc.) or α-2,3-Neuraminidase from Macrobdella decora expressed in Escherichia coli (ProZyme, Inc.) at 37 °C for 2 h. hPods were also digested with both Sialidase A™ and O-Glycanase™ from Streptococcus pneumoniae expressed in E. coli (ProZyme, Inc.) at 37 °C for 12 h to remove Core1-related O-glycans completely. Platelet aggregating activity was measured by WBA Carna (IMI, Saitama, Japan) as described previously (31Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain.Biochem. Biophys. Res. Commun. 2006; 349: 1301-1307Crossref PubMed Scopus (171) Google Scholar). Heparinized mouse whole blood was drawn from BALB/c mice. Two hundred microliters each of mouse whole blood samples and NZ-1 or control rat IgG in four reaction tubes was stirred at 1,000 rpm at 37 °C, and pre-incubated for 2 min, followed by the addition of 12 μl each of cells (2 × 107 cells/ml). One to five minutes later, whole blood samples were sucked to detect aggregation pressure at a rate of 200 μl/6.4 s using a syringe containing screen microsieves made of nickel, 3.7 mm in diameter, with 300 openings of 20 × 20 μm2 in a 1-mm diameter area. The final platelet aggregation pressure of each reaction tube was determined at the pressure rate (%) of a pressure sensor connected to the syringe. To achieve extremely high throughput and feasibility, the antibody-overlay lectin microarray is considered to be an ideal approach to achieve differential glycan analysis targeting a particular glycoprotein, by using a lectin microarray infrastructure (21Rosenfeld R. Bangio H. Gerwig G.J. Rosenberg R. Aloni R. Cohen Y. Amor Y. Plaschkes I. Kamerling J.P. Maya R.B. A lectin array-based methodology for the analysis of protein glycosylation.J. Biochem. Biophys. Methods. 2007; 70: 415-426Crossref PubMed Scopus (95) Google Scholar). From a practical viewpoint, there is a critical issue to be overcome when using antibodies for glycoprotein detection, since the antibody glycans potentially interact with lectins immobilized on the glass slide (Fig. 2A, top). To examine this possibility, we first carried out a pilot experiment of the antibody-overlay lectin microarray using hTf, which mainly contained sialylated biantennary, complex-type N-glycans at two N-glycosylation sites (22Kuno A. Uchiyama N. Koseki-Kuno S. Ebe Y. Takashima S. Yamada M. Hirabayashi J. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.Nat. Methods. 2005; 2: 851-856Crossref PubMed Scopus (439) Google Scholar), as a model glycoprotein. Lectin microarray comprising 43 lectins (supplemental Fig. S1) was incubated first with or without 1 ng of hTf and then with 20 ng of biotinylated rabbit anti-hTf polyclonal antibody followed by Cy3-streptavidin detection as described under “Experimental Procedures”. Signals were observed both in the presence and absence of hTf on extensive spots of α2–6 Sia-binders, SSA, SNA, and TJA-I, as well as a β-Gal-binder, RCA120 (Fig. 2B). This indicates a typical glycan profile to serum antibody, i.e. partially α2–6 sialylated biantennary complex type N-glycan. In fact, positive signal intensities (P) (in the presence of hTf) were almost comparable with those of negative ones (N) (in the absence of hTf), indicating the derived signals were not reliable. To suppress such undesirable background noise, attributed to the detection antibody, while preserving specific signals of hTf, effective blocking of the analyte (hTf)-bound microarray was attempted using an excess amount of non-labeled “human” polyclonal IgG (hpIgG) prior to detection by anti-hTf antibody (Fig. 2A, bottom). The blocker, hpIgG, having substantially the same glycoforms as those of the detection antibody (“rabbit” polyclonal antibody in this case), but lacking the epitope (biotin) for fluorescence detection, is expected to work for masking the residual binding sites on the array. Technical consideration was also taken to minimize the blocking time (30 min) to suppress the analyte exchange between the pre-captured analyte (hTf) and an excess amount of the blocker hpIgGs. However, no detectable signal reduction was observed even after 2 hours of incubation with the blocker hpIgG, as examined by direct probing of the array with Cy3-labeled hTf (Fig. 2C). As a result of blocking with 20 μg of hpIgG, the background signals totally disappeared in the absence of hTf, whereas intense (i.e. specific) signals were observed, e.g. on RCA120, in the presence of the analyte (Fig. 2B). The obtained signal pattern was closely correlated with the reported glycan structures of hTf (21Rosenfeld R. Bangio H. Gerwig G.J. Rosenberg R. Aloni R. Cohen Y. Amor Y. Plaschkes I. Kamerling J.P. Maya R.B. A lectin array-based methodology for the analysis of protein glycosylation.J. Biochem. Biophys. Methods. 2007; 70: 415-426Crossref PubMed Scopus (95) Google Scholar). To profile a relatively small amount (<100 ng) of a target glycoprotein with the aid of antibody-ov

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