Analysis of Glycosphingolipid Glycans by Lectin Microarrays

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Analysis of Glycosphingolipid Glycans by Lectin Microarrays Haoqi Du, Hanjie Yu, Tianran Ma, Fuquan Yang, Liyuan Jia, Chen Zhang, Jiaxu Zhang, Lili Niu, Jiajun Yang, Zhiwei Zhang, Kun Zhang, and Zheng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01945 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

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Analytical Chemistry

Analysis of Glycosphingolipid Glycans by Lectin Microarrays Haoqi Du1‡, Hanjie Yu1‡, Tianran Ma1, Fuquan Yang2, Liyuan Jia1, Chen Zhang1, Jiaxu Zhang1, Lili Niu2, Jiajun Yang1, Zhiwei Zhang1, Kun Zhang1 & Zheng Li1* 1Laboratory

for Functional Glycomics, College of Life Sciences, Northwest University, Xi’an, China. of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. 2Laboratory

‡These authors contributed equally to this work. *Zheng Li, Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, No. 229 Taibai Beilu, Xi’an 710069, China. Tel: +86-29-88304104; Fax: +86-29-88303572; E-mail: [email protected] ABSTRACT: Glycosphingolipids (GSLs) are ubiquitous glycoconjugates on cell membrane. Identification of unknown GSL-glycan structures is still a major challenge. To address this challenge, we developed a novel strategy for analysis of GSL-glycans from cultured cells based on a lectin microarray that could directly detect and reveal glycopatterns of GSL extracts without the need for glycan release. There were six steps to perform analysis of GSL-glycans: (i) extraction of GSLs from cell pellets, (ii) quantification of GSL-glycans using orcinol-sulphuric acid reaction, (iii) lyso-GSLs were prepared by using the sphingolipid ceramide N-deacylase, (iv) fluorescence labeling of lyso-GSLs, (v) detection by a lectin microarray, (vi) data acquisition and analysis. Simultaneously, a supplementary verification analysis for GSL-glycans was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. An optimized experimental condition, which consist of the blocking buffer, incubation buffer, and appropriate GSL concentration, was investigated by analyzing glycopatterns of a standard ganglioside (GM1a) via a lectin microarray. The results of GSL-glycans from human hepatocarcinoma cell lines (MHCC97L, MHCC97H, and HCCLM3) showed there were 27 lectins (e.g. WFA, MAL-II, and LTL) to give significantly differential signal compared with normal human liver cell line (HL-7702), indicating up and/or down regulations of corresponding glycopatterns such as α1-2 fucosylation and α2-3 sialylation, and changes of certain glycostructures such as Galβ1-3GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc:Cer and GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ14Glc:Cer. The lectin microarray analysis of lyso-GSLs labeled by fluorescence has proven to be credible, which could provide the glycopatterns and detailed linkage information of GSL-glycans.

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates on cell membrane, which are minor components of cell membranes and are also major constituents of endoplasmic reticulum membrane compartments and neuronal cell membranes. GSLs are consist of a glycan structure extending into the extracellular matrix and a lipid moiety anchoring into the outer leaflet of cell membranes. This combination makes GSL an amphiphilic molecule, and it is highly related to various biological processes such as cell signaling, proliferation, adhesion, apoptosis, and oncogenesis.1-3. Over the past years, the mass spectrometry (MS)-based glycosphingolipidomics have become emerging technologies for the structural analysis of GSLs4-7. These methods include (i) classical thin-layer chromatography (TLC)8; (ii) liquid-based separation techniques such as high performance liquid chromatography (HPLC) and hydrophilic interaction liquid chromatography9; (iii) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS10; (iv) the on- and off-line capillary electrophoresis MS11,12; (v) ion mobility -MS systems13. Recent developments in MS-based

technologies have provided a valuable and extensible insight into the field of glycomics as well as lipidomics of heterogeneous GSL mixtures by the combination of efficient separation techniques and high resolution MS. However, the complexities of GSL biosynthesis, structure, and function, especially the physicochemical diversity and variability increase the difficultly of GSL structural analysis4. Furthermore, the function of different GSL glycosyl epitopes might be completely opposite in tumor and other diseases, some promote the progress of disease, while some others inhibit14,15. Consequently, the glycan compositional elucidation of GSL extracts from complex biological or clinical samples is crucial to researchers to explore the structures and the further biological mechanisms of GSLs. Lectins are natural substances mostly originated from plant, which can be utilized in the investigations to detect and reveal carbohydrate structures of glycoconjugates16. The emergence of lectin-related high-throughput glycomic techniques such as lectin microarrays made it possible to explore multiple, distinct binding interactions simultaneously. And lectin microarrays

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0.1 M of sodium hydroxide. Sep-Pak C18 cartridges were used for the purification of GSLs. Sample Preparation. SCDase de-N-acylated derivative of GM1a (Lyso-GM1a) and SCDase de-N-acylated derivative of cellular GSLs (lyso-GSLs) were prepared under the treatment of SCDase under the manufacturer’s recommendation with minor modifications23. Briefly, GSLs were resuspended in 35 mM of sodium acetate buffer (pH 5.8) containing 100 mM of calcium chloride, 0.28% TDC followed by the employment of 20 mU of SCDase at 37 °C for 16 h. Lyso-GSLs were purified with Sep-Pak C18 cartridges and then labeled with AmershamTM Cy3 Mono-Reactive Dye under the manufacturer’s recommendation. The redundant Cy3 was removed with Sephadex G-25 cartridges. Investigation of Experimental Conditions for Lectin Microarrays Analyzing GSL-glycans. Ganglioside GM1a as a standard GSL that was used to test the reliability of lectin microarrays and to optimize the experimental conditions for lectin microarrays analyzing GSL-glycans. Negative tests, the optimization of blocking buffer, incubation buffer, incubation time, and GSL-glycan concentration were carried out to achieve the final experimental conditions for lectin microarrays in further studies. TLC analyses of GM1a, lyso-GM1a, and lysoGM1a labeled with Cy3 were carried out to evaluate the reliability of SCDase lyso-derivatives and Cy3 labeling. Lectin Microarrays. Lectin microarrays were prepared and carried out according to the protocol with modifications16. The layout of a lectin microarray are shown in Figure 2A. Each lectin was spotted in triplicate per block with triplicate blocks on one slide (Figure S1). After immobilization, the lectin microarrays were then subjected to the analysis of GSLglycans. The lectin microarrays were blocked with the blocking buffer containing 50 mM MEA in 50 mM sodium borate for 1 h. The appropriate amount of Cy3-labeled lyso-GSLs were diluted in 0.5 mL of incubation buffer containing 2% BSA, 1.5 M glycine, 0.1% Tween-20, and 0.06% TritonX-100 in 1 × PBS, and then applied to the blocked lectin microarrays, followed by the incubation in the incubation chamber at 37 °C for 3 h in a rotisserie oven set at 5 rpm. Each sample was consistently observed from three repeated slides. The normalized data of the testing group and the control group were then compared to determine any relative change in GSL glycosylation levels. Glycan Release from GSLs. Glycan release was carried out according to previous report with slight modifications25. Briefly, dried GSL extracts were added with 150 μL of 6% NaClO under stirring, after 30 min in room temperature, 60 μL of octanol and 10 μL of formic acid were added carefully on ice. After that, the mixture was stirred until no bubble can be observed and stored at 4 °C overnight. The mixture was centrifuged for 10,000 rpm, 10 min and the upper layer was removed. 0.5 mL of decane was used twice to remove fatty residues. GSL-glycans were purified by HyperSepTM HypercarbTM SPE cartridges. MALDI-TOF/TOF-MS Analysis for GSL-Glycans. The purified GSL-glycans were subjected to a Bruker Daltonics’ UltrafleXtremeTM MALDI-TOF/TOF mass spectrometer according to previous reports26,27. GSL-glycans were evaporated and redissolved in 5 μL of water, and 1 μL was spotted on an MTP AnchorChip var/384 sample target and

have become a primary method to investigate glycosylation of entire samples. Easy sample preparation, and simultaneous quantitative analysis of N- and O-linked glycans on intact biological structures can be achieved. This is the most remarkable prerogatives of lectin microarrays, which guarantees the real state of protein glycosylation in sample being correctly reflected16-20. In this study, we developed a novel method that could directly detect and reveal glycopatterns of GSL extracts by a lectin microarray without the need for glycan release from complex biological or clinical samples (Table S1). There were six steps to perform analysis of GSL-glycans: (i) extraction of GSLs from cell pellets21,22; (ii) quantification of GSL-glycans using orcinol-sulphuric acid reaction; (iii) preparation of lyso-GSLs by using the sphingolipid ceramide N-deacylase (SCDase)23,24; (iv) fluorescence labeling of lyso-GSLs; (v) detection by a lectin microarray (Figure 1A); (vi) data acquisition and analysis. Simultaneously, a supplemental verification analysis for GSL-glycans was performed by MALDI-TOF MS. Finally, a comprehensive annotation based on the results of the lectin microarrays and MALDI-TOF MS present the real glycan structures containing different linkages of GSLs (Figure 1B). EXPERIMENTAL SECTION Materials and Instrument. Ganglioside GM1a, sodium acetate, sodium taurodeoxycholate (TDC), monoethanolamine (MEA), sodium borate, Tween-20, BSA, glycine, TritonX-100, octanol, decane, and 2,5-dihydroxybenzoic acid were purchased from Sigma-Aldrich. SCDase was purchased from Takara. RPMI-1640 medium, high-glucose DMEM medium, AmershamTM Cy3 Mono-Reactive Dye Pack and SepadaxTM PD MiniTrap G-25 columns were purchased from GE Healthcare. Sep-Pak C18 cartridges were purchased from Waters. Fetal bovine serum (FBS), penicillin, streptomycin, and HyperSepTM HypercarbTM SPE cartridges were purchased from Thermo Scientific. Sodium hypochlorite and formic acid were purchased from J&K Scientific. 37 lectins were purchased from Sigma-Aldrich and Vector Laboratories. TLC LuxPlate Silica gel 60 plates and calcium chloride were purchased from Merck. Spotting of lectin microarrays was carried out on a SmartArrayer 48 Microarray Spotter (CapitalBio) with SMP10B Stealth microarray spotting pins (Arrayit). Scan of lectin microarrays was performed on a GenePix 4100A Microarray Scanner (Molecular Devices). Mass spectrometric analysis was performed on an UltrafleXtremeTM MALDI-TOF/TOF mass spectrometer (Bruker Daltonics). Cell Culture. HL-7702 cells were cultured in RPMI-1640 medium, containing 10% (v/v) fetal bovine serum FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. MHCC97L, MHCC97H and HCCLM3 cells were cultured in high-glucose DMEM medium and supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were grown with 15 mL of medium at 37 °C under a 5% CO2 humidified atmosphere in T-75 flasks. Extraction of GSLs. The GSLs were extracted according to the protocol with minor modifications7,21,22. Briefly, cells were cultured until they reached ~100% confluency. Total lipids were extracted by homogenization on ice using an ultrasonic homogenizer for three stages with the solvent of chloroform/methanol (2/1 (v/v) for stage i, 1/1 (v/v) for stage ii, and 1/2 (v/v) for stage iii). Phospholipids were eliminated with


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Analytical Chemistry appropriate sample concentration in our lectin incubation system. Furthermore, negative tests confirmed the reliability of lectin microarrays analyzing GSL-glycans. All scan results (Figure S5), including results of blank microarray after produced and rinsed, results of microarray after blocking in blocking buffer for 1 h, results of microarray after incubation with Cy3 fluorescent dye in incubation buffer for 3 h, and results of microarray after incubation with non-labeled lyso-GSL in incubation buffer for 3 h, showed no significant signal expect for the positive control marker. In conclusion, the conditions of analyzing GSL-glycans via lectin microarrays were 50 mM MEA in 50 mM borax (pH 7.4) as the blocking buffer, PBS containing 0.06% Triton X-100, 0.1% Tween-20, 2% BSA and 1.5 M glycine as the incubation buffer, 3 hours of incubation time, and 0.05 μg/μL of GSLglycans as the appropriate sample concentration. Lectin Microarray Analysis of GSL-Glycans. To evaluate the applicability of our method for the comprehensive analysis of the real glycan structures containing different linkages of GSLs from complex samples. we applied our method to analyze the glycopatterns of GSLs from normal human liver cell (HL7702) and three human hepatocarcinoma (HCC) cell lines (MHCC97L, MHCC97H, and HCCLM3) with different metastasis potentials using the lectin microarrays. The layout of the lectin microarrays and glycopatterns of Cy3-labeled lysoGSLs from normal liver and HCC cells bound to the lectin microarrays were shown in Figure 2A. The normalized fluorescent intensities (NFIs) for each lectin were summarized as the mean values ± SD in Table S2. Lectin signal patterns were classified into three categories to evaluate whether the glycopatterns of GSLs were altered between HCC lines and normal control: (i) results showing significant increases in NFIs (fold-change ≥ 1.50, P < 0.05), (ii) results showing significant decreases in NFIs (fold-change ≤ 0.67, P < 0.05), and (iii) results showing almost even level in NFIs (fold-change range from 0.67 to 1.50, no significant difference). All based on fold change in pairs (with p-values lower than 0.05) with the NFIs of each lectin from normal liver and liver cancer cells were shown in Table S3. To verify the repeatability of lectin microarrays, a hierarchical clustering analysis was performed by importing the generated data from three biological replicates into EXPANDER 6.0 software (Figure 2B). To sum up, there were 27 lectins to give significantly differential signal from normal liver cell line and HCC cells. The results showed that the Siaα2-3Gal binder MAL-II, α1-3/1-6Man and Galα13Galβ1-4GlcNAc binder HHL, Fucα1-2Galβ1-4GlcNAc binder LTL, Fucα1-4GlcNAc NPA, GalNAcα1-3Gal and GalNAcα1-3Galβ1-3/1-4Glc binder PTL-I, terminal GalNAc/Gal binder SJA, αGalNAc and GalNAcα1-3(Fucα1-2) Gal binder DBA, Bisecting GlcNAc binder PHA-E, Galβ13GalNAc, GlcNAcα1-3/1-4Gal, and GalNAcβ1-4GlcNAc binder PNA, Galβ1-3GalNAc/GlcNAc and GalNAc binder MPL, GalNAc binder GSL-I, β-GlcNAc and Galβ1-4GlcNAc binder DSA, Sia2-3Gal and Galβ1-4GlcNAc binder MAL-I, and α1-3Man, Galα1-3Galβ1-4GlcNAc binder GNA exhibited increased NFIs in HMCC97L compared with HL-7702 (all fold change ≥ 1.54, P < 0.05), on the other hand, the terminal GalNAcα/β1-3/6Gal binder WFA, Fucα1-3Galβ1-4GlcNAc and Fucα1-6GlcNAc binder AAL, Fucα1-6GlcNAc binder

dried. Then 1 μL of 20 mg/mL 2,5-dihydroxybenzoic acid was spotted to recrystallize the GSL-glycans. Mass calibration was performed with the peptide calibration standards 250 calibration points (Bruker Daltonics). Measurements were taken in positive-ion and reflectron mode and the intense ions from mass spectra were subsequently selected and subjected to MS/MS. A total of 1500 laser shots per pixel (200 laser shots per position of a random walk within each pixel) were collected and the data were acquired using the Flex software suite (FlexControl 3.3, FlexAnalysis 3.3, Bruker Daltonics). The original intensity of each peak was analyzed by FlexAnalysis 3.3, and the relative intensities of each peak were calculated as a percentage of the total intensity of all glycan peaks in every group28,29.GSL-glycan structures were annotated by Glycoworkbench software30,31. By combining the information received from fragmentation of [M+H]+ and [M+Na]+ ions of the glycans from GSLs, a complete structural characterization in terms of linkage, branching of glycosidic bonds of oligosaccharides were achieved. RESULTS AND DISCUSSION Experimental Conditions for Lectin Microarrays. As shown in Figure S2A, lyso-GM1a with lower RF showed a lower band compared to the GM1a band, indicating that the lyso-GM1a digested by SCDase became more hydrophilic than the GM1a molecule. The slight rise of Cy3-GM1a band compared with lyso-GM1a band indicated that the Cy3 labeling was successful and efficient. As shown in Figure S2B, the emergence of the lyso-GM1a band suggested that after the SCDase treatment, sphingoid amino group was exposed to be detected by ninhydrin spray and Cy3 labeling was realizable. The Cy3-GM1a band became weaker indicated that some lysoGM1a molecules been labeled with Cy3 and their amino group has been blocked. The result of the lectin microarray analysis of Cy3-GM1a are shown in Figure S3. 27 of 37 lectins showed valid signals. Among these lectins, 9 lectins recognize structure Gal or GalNAc (Figure S3B), 2 lectins recognize structure Galβ1-4Glc (Figure S3C), moreover, the Siaα2-3Gal binder lectin MAL-II showed significant signal while the Siaα2-6Gal binder lectin SNA showed no significant signal (Figure S3D). Given that Gal/GalNAc, Galβ1-4Glc, and Siaα2-3Gal are mean structures of a GM1a molecule, the result of lectin microarrays was reliable. As shown in Figure S4, the optimization of the experimental conditions containing blocking buffer (Figure S4A), incubation buffer (Figure S S4B), incubation time (Figure S4C), and GSLglycan concentration in the incubation system (Figure S4D). The result of the optimization of blocking buffer showed that MEA was a better blocking buffer than BSA, which could give lectins stronger signals after incubation. The result of the optimization of blocking buffer showed that the introduction of 0.06% TritonX-100 into the usual incubation buffer of lectin microarrays leaded to stronger lectin signals by facilitating the hydrophilized GSL-glycans bind more compact with lectins, while TDC showed no significant effect on the binding between GSL-glycans and lectins. The result of the optimization of incubation time showed that 3 hours was enough for the bindings between GSL-glycans and lectins. The result of the optimization of GSL-glycan concentration in the incubation system showed that 0.05 μg/μL of GSL-glycans was the



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identified and annotated with proposed structures from HL7702, MHCC97L, MHCC97H, and HCCLM3 cell lines according to the results of lectin microarrays, respectively. Of these, there were 6 glycan signals (m/z 728.211 [Galβ1-3Galα14Galβ1-4Glc], 747.256 [Galβ1-3GlcNAcβ1-3Galβ1-4Glc], 876.298 [GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc], 893.314 [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc and/or Fucα12Galβ1-3GlcNAcβ1-3Galβ1-4Glc], 1038.351 [Galβ13GalNAcβ1-4(NeuAcα2-3)Galβ1-4Glc], and 1055.366 [Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc] that to be present in all cell lines with different intensities. 9 glycan signals (m/z 980.309 [NeuGcα2-8NeuAcα2-3Galβ1-4Glc], 1096.393 [GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ14Glc], 1112.388 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ13Galβ1-4Glc], 1134.370 [Galβ1-3GlcNAcβ1-3Galβ13GlcNAcβ1-3Galβ1-4Glc], 1201.424 [Galα1-3(Fucα12)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc], 1242.451 [GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ14Glc], 1286.348 [NeuAcα2-3SulfGalβ1-4(Fucα13)GlcNAcβ1-3Galβ1-4Glc], 1550.562 [Fucα1-2Galβ14(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ14Glc], and 1712.614 [Galα1-3(Fucα1-2)Galβ1-4(Fucα13)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc]) were present only in HCC cell lines, 7 glycan signals (m/z 1096.393 [GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-3Galβ14Glc], 1112.388 [Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ13Galβ1-4Glc], 1201.424 [Galα1-3(Fucα1-2)Galβ1-4(Fucα13)GlcNAcβ1-3Galβ1-4Glc], 1242.451 [GalNAcα1-3(Fucα12)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc], 1286.348 [NeuAcα2-3SulfGalβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc], 1550.562 [Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ14(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc], and 1712.614 [Galα13(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα13)GlcNAcβ1-3Galβ1-4Glc]) were present only in MHCC97H and/or HCCLM3 cell lines with high metastasis potential. There was a signal at m/z 1280.428 [Galβ1-3GlcNAcβ1-3Galβ13(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc] that was unique in HL7702 cell line, 1 glycan signal (m/z 1134.370 [Galβ13GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc]) was unique in MHCC97L cell line with low metastatic potential, and 5 glycan signals (m/z 1096.393 [GalNAcα1-3(Fucα1-2)Galβ13GlcNAcβ1-3Galβ1-4Glc], 1112.388 [Galβ1-3GlcNAcβ13Galβ1-3GlcNAcβ1-3Galβ1-4Glc], 1201.424 [Galα1-3(Fucα12)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc], 1242.451 [GalNAcα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ14Glc], and 1286.348 [NeuAcα2-3SulfGalβ1-4(Fucα13)GlcNAcβ1-3Galβ1-4Glc]) were unique in HCCLM3 cell line with high metastatic potential. The MALDI-TOF/TOF-MS of the precursor ions m/z 893.314, 1038.351, 1055.366, and 1712.614 were shown in Figure. 4. The results showed that due to low energy was needed, the major fragment ion B- and Y-type cleavages could provid detailed sequencing information. The cross-ring fragment ions that provide composition and linkage information were also detected in the MALDI-TOF/TOF mass spectra34. For example, the fragment ions B2Z3 (348.129), and B1 (747.256) in the m/z 893.314, 0,4X1Z4 (831.277), and Y4 (909.309) in the m/z 1055.366, B2Y6 (325.113), Z3Y6 (1386.493), and Y3 (1566.557) in the m/z 1712.614 elucidated the occurrence of Fuc. Y3Z3 (364.113) indicated that the occurrence of Neu5Ac in the m/z 1038.351. The cross-ring fragment ions 1,4X3 (803.282)

LCA, terminal GlcNAc/Gal binder ConA, GalNAc, GalNAcβ13Gal and Galβ1-3/1-4GlcNAc binder VVA, and Galβ13GalNAc binder ACA showed significant decreased NFIs in HMCC97L compared with HL-7702 (all fold change ≤ 0.63, P < 0.001). Similarly, there were 13 lectins (e.g., PSA, PTL-I, and MAL-II) that showed significant increased NFIs (all fold change ≥ 1.55, P < 0.05), and 5 lectins (e.g., ECA, LCA, and AAL) that showed significant decreased NFIs (all fold change ≤ 0.60, P < 0.001) in HMCC97H compared with HL-7702. And there were 11 lectins (e.g., SJA, SBA, and PNA) that showed significant increased NFIs (all fold change ≥ 1.51, P < 0.01), and 7 lectins (e.g., WFA, GSL-II, and NPA) that showed significant decreased NFIs (all fold change ≤ 0.60, P < 0.01) in HCCLM3 compared with HL-7702 (Figure S6). As shown in Figure 2C, the Fucα1-4GlcNAc binder NPA, showed significant increased NFIs in HMCC97L compared with HL-7702 (fold change = 1.55, P < 0.001). The Siaα2-3Gal binder MAL-II, GalNAc, GalNAcα1-3Gal, and GalNAcα13Galβ1-3/1-4Glc binder PTL-I, as well as GalNAc, Galβ13GalNAc, and Galβ1-3GlcNAc binder MPL showed significant increased NFIs in HMCC97L and HMCC97H compared with HL-7702 (all fold change ≥ 2.02, P < 0.05). The Fucα1-2Gal and Fucα1-3GlcNAc binder LTL as well as αGalNAc and GalNAcα1-3(Fucα1-2)Gal binder DBA showed significant increased NFIs in HMCC97L, HMCC97H, and HCCLM3 compared with HL-7702 (all fold change ≥ 1.55, P < 0.05), and significant increased NFIs in HCCLM3 compared with HMCC97L (all fold change ≥ 1.40, P < 0.05). The αGalNAc binder GSL-I showed significant increased NFIs in HMCC97L and HCCLM3 compared with HL-7702 (all fold change ≥ 2.50, P < 0.001). The Fucα1-3Galβ1-4GlcNAc and Fucα1-6GlcNAc binder AAL showed significant decreased NFIs in HMCC97L, HMCC97H, and HCCLM3 compared with HL-7702 (all fold change ≤ 0.54, P < 0.01). The idea that using (neo)glycolipid microarray into glycomic research32,33 and the characters of lectin gave us the inspiration to develop this method. In consideration of the harsh alkaline conditions required for GSL de-N-acylation and the need for the labeling of Cy3 dye, we chose the SCDase method for processing original GSL molecules extracted from cultured cells, which was commercially available and of high catalytic efficiency and broad fatty acid specificity23. Furthermore, instead of using expensive enzymes such as endoglycoceramidase to prepare GSL-glycan for MS analysis, we chose oxidative release of GSL-glycan, which was low cost and easy to conduct. The investigation of the detection limits of lectin microarrays showed with the increasing abundance of GSL-glycans, detectable and valid fluorescence intensities emerged at 0.033 μg/μL of GSL-glycan concentration, and then stabilized at an optimal concentration of 0.050 μg/μL. Hence, a relatively small amount of abundance of GSL-glycans (25 μg) was needed for a microarray experiment. MALDI-TOF/TOF-MS Analysis of GSL-Glycans. To obtain complementary information of GSL-glycans from normal human liver cell and three HCC cell lines with different metastasis potentials, we utilized the NaClO oxidative release of GSL-glycans25, and analyzed GSL-glycans by MALDITOF/TOF MS. The results were shown in Figure 3 and Table S4. A total of 11, 11, 11, and 15 GSL-glycan signals were


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Analytical Chemistry efficiency46. In the last decade, the advent of ion mobility spectrometry with MS allows the detection of both structural and mass information for ions could be reached within milliseconds, developing into a new technique for the investigation of small molecule13, and it has been proven to be a effective and powerful analysis approach that could detect and analyze glycolipid species with potential biomarker role unambiguously9. However, the complexity of glycomes, as well as the number of glycan determinants47 still remain a big challenge to glycoscientists despite the rapid emergence of techniques in the field of MS and other analysis instrument. There are more than 3,700 different existing GSL structures were included in the human GSL metaglycome, when the tremendous diversity of the lipid moiety of GSL was taken into consideration, according to the Lipid Maps Database48,49. Although they share a lactosylceramide-type glycan sturcture as the non-reducing sugar, it is still a vast task to isolate and identify every single GSL from such a large-scale repertoire. The main purpose of this study is to establish a facile method to conduct glycolipidomics analyses in a more time-saving approach and from the perspective of lectins with lower cost. And by using lectin microarray to analyze GSL-glycans, there will be no demand of separating certain GSL by traditional methods such as HPLC due to the nature of high-throughput omics-level screening of lectin microarrays, the whole process of sample preparation was easier to be achieved. CONCLUSIONS We developed a novel strategy for comprehensive analysis of GSL-glycans from cultured cells based on lectin microarrays that allows the simultaneous large-scale profiling and monitoring of GSL-glycans in a single method, with the supplementary data from MALDI-TOF/TOF-MS analysis, application of this strategy enabled the identification of exact structures of GSL-glycans from various biological samples to provide more target and insight for the diagnosis and treatment of diseases in the challenging work of GSL analysis. Analyzing neutral or acidic GSLs respectively using this strategy is also realizable by simply separating neutral/acidic GSLs when extracting GSL from biological samples. Our method could provide detailed information of glycan linkages from the perspective of lectins with lower cost, high-throughput and easy to conduct.

indicated that the Fuc was 4-linked to the GlcNAc, the crossring fragment ions C31,4X3 (722.235) indicated a 2-3 linkage between Neu5Ac and Gal, the cross-ring fragment ions 0,2X3Y5 (1138.404) indicated that the Fuc was 3-linked to the GlcNAc. It is reported that the fucosylation of glycan is one of the many biological phenomena during the tumorgenesis including HCC and it has an outstanding potential of being a biomarker as well as target in terms of the diagnosis and treatment of cancer35-37. In this study, NFIs of LTL showed a significant up-regulation in all HCC cells compared to normal control, which indicated that the abundance of GSLs with the stricture Fucα1-2Gal were up-regulated during the development of HCC, and so as the metastasis ability of HCC cell rise. Combining the results of lectin microarrays and MALDI-TOF MS, GSL structures such as Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc:Cer, Fucα12Galβ1-3GlcNAcβ1-3Galβ1-4Glc:Cer, GalNAcα1-3(Fucα12)Galβ1-3GlcNAcβ1-3Galβ1-4Glc:Cer are likely to be upregulated in HCC cells. The results of lectin MAL-II and MS analysis indicated that gangliosides containing Siaα2-3Gal such as GM1 and GM2 were up-regulated during the development of HCC, which could be a antitumoric target38. GM2 outexpressed than normal control in human breast cancer stem cells39 could also be used as target in HCC treatment. GM3 was reported to be suppressant of cancer development and progression by the inhibitory effect on activation of growth factor receptors40,41. In this study, no GM3 was detected in the MS analysis. The NFIs of poly-sialic acid binder lectin WGA showed no significant difference indicated that GSL-glycans containing terminal Siaα2-8 remained unchanged. The upregulation of globo-series GSL was previously reported in breast cancer tissues42 and we found similar results in HCC cell lines. With the rapid development and evolution of modern instruments during the few decades, many breakthroughs have been made among glycolipidomics studies by high-end analytical platforms ranging from TLC, HPLC, to MS-based methods including but not limited to ion mobility mass spectrometry4, MALDI-quadrupole ion trap-TOF MS43, chipbased nanoelectrospray quadrupole-TOF-MS44, and 45 nanoelectrospray Orbitrap MS . Ion mobility spectrometry is a gas-phase electrophoretic technique that can separate ions according to their size, shape and charge state with high



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FIGURES

Figure 1. Flow chart of the comprehensive analysis of GSL-glycans by lectin microarrays and MALDI-TOF MS and glycopatterns of GSLglycans from liver cell lines. (A) Workflow of lectin microarray analysis of GSL-glycans. (B) Workflow of detection of GSL-glycans using MALDI-TOF-mass spectrometry.


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Figure 2. Glycopatterns of GSL-glycans from HL-7702, HMCC97L, HMCC97H, and HCCLM3 cell lines. (A) Layout of a lectin microarray, and Glycopatterns of Cy3-labeled GSL-glycans from HL-7702, HMCC97L, HMCC97H, and HCCLM3 cell lines. Several lectins showed significant signal differences were highlighted with orange frames. (B) Heat map and hierarchical clustering analysis of the 37 lectins with three biological replicates. Lectins were listed in rows, samples were listed in columns. The color and intensity of each square indicated expression levels relative to other data in the row. Red, high; green, low; black, medium. (C) Alterations of GSL glycopatterns between HCC groups with normal control in terms of lectins correlated to the sialylated, fucosylated, and GalNAc in GSL-glycans. *P < 0.05; **P < 0.01; ***P < 0.001. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; Man, green circle.


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Figure 3. MALDI-TOF mass spectra of GSL-glycans identified and annotated with proposed structures according to the results of lectin microarrays. Spectra of GSL-glycans from (A) HL-7702 cell line, (B) MHCC97L cell line, (C) MHCC97H cell line, and (D) HCCLM3 cell line. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; NeuGc, white diamond. CG, cyanomethyl glycosides residue.

Figure 4. MALDI-TOF/TOF-MS analysis of GSL-glycan precursor ions in mass spectra. Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Four major GSL-glycan peaks are indicated: (A) m/z 893.314, (B) m/z 1038.351, (C) m/z 1055.366, and (D) m/z 1712.614. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; CG, cyanomethyl glycosides residue.

ASSOCIATED CONTENT

AUTHOR INFORMATION

Supporting Information

Corresponding Author

Supplementary experimental section, (Extraction of GSLs from Cell Pellets, Purification of GSLs, Thin-layer Chromatography, Purification of GSL-Glycans); Supplementary figures, (Figure S1. Design and layout of a lectin microarray. Figure S2. TLC analysis of GM1a, lyso-GM1a, and Cy3-GM1a. Figure S3. Lectin microarray analysis of GM1a. Figure S4. The optimization of lectin microarrays analysis of GM1a. Figure S5. Negative tests of lectin microarrays. Figure S6. Glycoprofiling of GSL-glycans from HCC and normal cell lines.); Supplementary tables, (Table S1. Time frame and major reagent consumption of the comprehensive analysis of GSL-glycans in this study. Table S2. Glycopattern in HL-7702, MHCC97L, MHCC97H, and HCCLM3 cell lines by the lectin microarray analysis based on data of 37 lectins giving significant signal. Table S3. Fold changes of glycopatterns of GSLs from MHCC97L, MHCC97H, and HCCLM3 cell lines compared with HL-7702 cell line, Table S4. Proposed structures for GSLglycans from HCC and HL-7702 cell lines based on MALDITOF/TOF-MS and lectin microarrays.) (PDF)

*Zheng Li, Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, No. 229 Taibai Beilu, Xi’an 710069, China. Phone: +86-29-88304104; Fax: +86-29-88303572; E-mail: [email protected]

Author Contributions All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China. (No. 81372365 and No. 81871955)

REFERENCES



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Figure 1. Flow chart of the comprehensive analysis of GSL-glycans by lectin microarrays and MALDI-TOF MS and glycopatterns of GSL-glycans from liver cell lines. (A) Workflow of lectin microarray analysis of GSLglycans. (B) Workflow of detection of GSL-glycans using MALDI-TOF-mass spectrometry. 263x114mm (150 x 150 DPI)

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Figure 2. Glycopatterns of GSL-glycans from HL-7702, HMCC97L, HMCC97H, and HCCLM3 cell lines. (A) Layout of a lectin microarray, and Glycopatterns of Cy3-labeled GSL-glycans from HL-7702, HMCC97L, HMCC97H, and HCCLM3 cell lines. Several lectins showed significant signal differences were highlighted with orange frames. (B) Heat map and hierarchical clustering analysis of the 37 lectins with three biological replicates. Lectins were listed in rows, samples were listed in columns. The color and intensity of each square indicated ex-pression levels relative to other data in the row. Red, high; green, low; black, medium. (C) Alterations of GSL glycopatterns between HCC groups with normal control in terms of lectins correlated to the sialylated, fucosylated, and GalNAc in GSL-glycans. *P < 0.05; **P < 0.01; ***P < 0.001. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; Man, green circle. 259x361mm (150 x 150 DPI)



Figure 3. MALDI-TOF mass spectra of GSL-glycans identified and annotated with proposed structures according to the results of lectin mi-croarrays. Spectra of GSL-glycans from (A) HL-7702 cell line, (B) MHCC97L cell line, (C) MHCC97H cell line, and (D) HCCLM3 cell line. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; NeuGc, white diamond. CG, cyanomethyl glycosides residue. 243x278mm (150 x 150 DPI)


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Figure 4. MALDI-TOF/TOF-MS analysis of GSL-glycan precursor ions in mass spectra. Precursor ions were subjected to MS/MS analysis to obtain cleavages, including glycosidic cleavages and cross-ring cleavages. Four major GSL-glycan peaks are indicated: (A) m/z 893.314, (B) m/z 1038.351, (C) m/z 1055.366, and (D) m/z 1712.614. Glc, blue circle; GlcNAc, blue square; Gal, yellow circle; GalNAc, yellow square; Fuc, red triangle; NeuAc, purple diamond; CG, cyanomethyl glycosides residue. 466x289mm (150 x 150 DPI)


Analysis of Glycosphingolipid Glycans by Lectin Microarrays

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