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N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

2016; Elsevier BV; Volume: 15; Issue: 1 Linguagem: Inglês

10.1074/mcp.m115.051235

ISSN

1535-9484

Autores

Stephanie Holst, Anna J.M. Deuss, Gabi W. van Pelt, Sandra J. van Vliet, Juan J. García‐Vallejo, Carolien A. M. Koeleman, André M. Deelder, Wilma E. Mesker, Rob A.�E.�M. Tollenaar, Yoann Rombouts, Manfred Wuhrer,

Tópico(s)

Galectins and Cancer Biology

Resumo

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation. Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation. Colorectal cancer (CRC) 1The abbreviations used are:CRCcolorectal cancerDHB2,5-dihydroxybenzoic aciddHexdeoxyhexoseDMEMDulbecco's Modified EagleFFuc, fucoseFCSfetal calf serumGalNAcN-acetylgalactosamineGlcNAcN-acetylglucosamineGuHClguanidine hydrochlorideHHex, hexoseNHexNAc, N-acetylhexosamineHILIChydrophilic interaction liquid chromatographyLC-ESI-MSliquid chromatography-electrospray ionization-mass spectrometryMALDI-TOF-MSmatrix-assisted laser desorption/ionization time-of-flight mass spectrometryMS/MStandem mass spectrometryNeuAcN-acetylneuraminic acidPCAprincipal component analysisPNGase Fpeptide N-glycosidase FRPMIRoswell Park Memorial InstituteSPEsolid phase extraction. is a very prevalent and heterogeneous pathology with highly variable disease progression and clinical outcome among patients. It is the third most common cancer in men and the second most common in women (1.Ferlay J. S. I. Ervik M. Dikshit R. Eser S. Mathers C. Rebelo M. Parkin D.M. Forman D. Bray F. Cancer incidence and mortality worldwide: IARC CancerBase No. 11. International Agency for Research on Cancer, Lyon, France2013Google Scholar) with a highly stage-specific patient survival (2.SEER Cancer Statistics Review, 1975–2011.in: Howlader N.N.A. Krapcho M. Garshell J. Miller D. Altekruse S.F. Kosary C.L. Yu M. Ruhl J. Tatalovich Z. Mariotto A. Lewis D.R. Chen H.S. Feuer E.J. Cronin K.A. National Cancer Institute, Bethesda, MD2013Google Scholar). Treatments are often curative for patients with local disease stages (stage I-II), whereas a 5-year survival of only 13% is observed in patients with distant metastasis (stage IV) (2.SEER Cancer Statistics Review, 1975–2011.in: Howlader N.N.A. Krapcho M. Garshell J. Miller D. Altekruse S.F. Kosary C.L. Yu M. Ruhl J. Tatalovich Z. Mariotto A. Lewis D.R. Chen H.S. Feuer E.J. Cronin K.A. National Cancer Institute, Bethesda, MD2013Google Scholar). As CRC is often asymptomatic in the first years, unfortunately, only 40% of the patients are diagnosed at stage I-II, thus pointing to the urgent need of sensitive diagnostic tools for early detection and consequently effective, curative treatment (3.Siegel R. Desantis C. Jemal A. Colorectal cancer statistics, 2014.CA Cancer J. Clin. 2014; 64: 104-117Crossref PubMed Scopus (2327) Google Scholar). In this context, understanding the complex mechanisms of CRC is an overriding condition for the development of new, more efficient means of detection, treatment, and prognosis of the disease. colorectal cancer 2,5-dihydroxybenzoic acid deoxyhexose Dulbecco's Modified Eagle Fuc, fucose fetal calf serum N-acetylgalactosamine N-acetylglucosamine guanidine hydrochloride Hex, hexose HexNAc, N-acetylhexosamine hydrophilic interaction liquid chromatography liquid chromatography-electrospray ionization-mass spectrometry matrix-assisted laser desorption/ionization time-of-flight mass spectrometry tandem mass spectrometry N-acetylneuraminic acid principal component analysis peptide N-glycosidase F Roswell Park Memorial Institute solid phase extraction. Altered glycosylation is a hallmark of cancer (4.Adamczyk B. Tharmalingam T. Rudd P.M. Glycans as cancer biomarkers.Biochim. Biophys. Acta. 2012; 1820: 1347-1353Crossref PubMed Scopus (382) Google Scholar) and is known to occur with cancer progression (4.Adamczyk B. Tharmalingam T. Rudd P.M. Glycans as cancer biomarkers.Biochim. Biophys. Acta. 2012; 1820: 1347-1353Crossref PubMed Scopus (382) Google Scholar, 5.Mechref Y. Hu Y. Garcia A. Hussein A. Identifying cancer biomarkers by mass spectrometry-based glycomics.Electrophoresis. 2012; 33: 1755-1767Crossref PubMed Scopus (114) Google Scholar) as glycans are involved in many cancer-associated events such as adhesion, invasion, and cell signaling (6.Peracaula R. Barrabés S. Sarrats A. Rudd P.M. de Llorens R. Altered glycosylation in tumours focused to cancer diagnosis.Dis. Markers. 2008; 25: 207-218Crossref PubMed Scopus (141) Google Scholar). As a result of altered glycan structures, cellular processes can be affected due to a change of interactions with glycan-binding proteins (7.Holst S. Wuhrer M. Rombouts Y. Chapter Six—Glycosylation characteristics of colorectal cancer.in: Richard R.D. Lauren E.B. Adv. Cancer Res. Academic Press, London, UK2015: 203-256Google Scholar, 8.Saeland E. van Kooyk Y. Highly glycosylated tumour antigens: Interactions with the immune system.Biochem Soc. Trans. 2011; 39: 388-392Crossref PubMed Scopus (6) Google Scholar, 9.de-Freitas-Junior J.C. Bastos L.G. Freire-Neto C.A. Rocher B.D. Abdelhay E.S. Morgado-Díaz J.A. N-glycan biosynthesis inhibitors induce in vitro anticancer activity in colorectal cancer cells.J. Cell. Biochem. 2012; 113: 2957-2966Crossref PubMed Scopus (26) Google Scholar). Several CRC tissue-associated changes in N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed (7.Holst S. Wuhrer M. Rombouts Y. Chapter Six—Glycosylation characteristics of colorectal cancer.in: Richard R.D. Lauren E.B. Adv. Cancer Res. Academic Press, London, UK2015: 203-256Google Scholar). For instance, N-glycans extracted from colorectal tumor tissues are characterized by an increase of sulfated glycans, (truncated) high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals (10.Balog C.I. Stavenhagen K. Fung W.L. Koeleman C.A. McDonnell L.A. Verhoeven A. Mesker W.E. Tollenaar R.A. Deelder A.M. Wuhrer M. N-glycosylation of colorectal cancer tissues: A liquid chromatography and mass spectrometry-based investigation.Mol. Cell. Proteomics. 2012; 11: 571-585Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In accordance, elevated expression of sialyl Lewis A (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R; NeuAc = N-acetylneuraminic acid, Gal = galactose, Fuc = fucose, GlcNAc = N-acetylglucosamine, R = rest) and pauci-mannosidic N-glycans (truncated high-mannose-type, Man1–4GlcNAc1–4GlcNAc; Man = mannose) was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas (11.Kaprio T. Satomaa T. Heiskanen A. Hokke C.H. Deelder A.M. Mustonen H. Hagström J. Carpen O. Saarinen J. Haglund C. N-glycomic profiling as a tool to separate rectal adenomas from carcinomas.Mol. Cell. Proteomics. 2015; 14: 277-288Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Due to limitations in accessibility of tumor materials and possibilities of in vivo studies on a large scale, cancer cell lines represent a relevant alternative and are widely used as model systems for studying the molecular mechanisms associated with cancer outcome and progression. Since the early 1960s, colorectal cancer cell lines have been established with HT29, LoVo, LS-180, LS-174T, and Co115 representing the first continuous cell lines derived from colon tumors and xenografts (12.Drewinko B. Romsdahl M.M. Yang L.Y. Ahearn M.J. Trujillo J.M. Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line.Cancer Res. 1976; 36: 467-475PubMed Google Scholar, 13.Tom B.H. Rutzky L.P. Jakstys M.M. Oyasu R. Kaye C.I. Kahan B.D. Human colonic adenocarcinoma cells. I. Establishment and description of a new line.In Vitro. 1976; 12: 180-191Crossref PubMed Scopus (381) Google Scholar, 14.von Kleist S. Chany E. Burtin P. King M. Fogh J. Immunohistology of the antigenic pattern of a continuous cell line from a human colon tumor.J. Natl. Cancer Inst. 1975; 55: 555-560Crossref PubMed Scopus (120) Google Scholar). Major benefits of cancer cell lines are their continuous availability, their fast growth, and relatively easy handling, making them suitable also for high-throughput screenings (15.Wilding J.L. Bodmer W.F. Cancer cell lines for drug discovery and development.Cancer Res. 2014; 74: 2377-2384Crossref PubMed Scopus (250) Google Scholar) and a large range of experimental possibilities (16.Rockwell S. In vivo-in vitro tumour cell lines: Characteristics and limitations as models for human cancer.Br. J. Cancer Suppl. 1980; 4: 118-122PubMed Google Scholar). Of note, advantages and limitations of cell lines have been recently reviewed (15.Wilding J.L. Bodmer W.F. Cancer cell lines for drug discovery and development.Cancer Res. 2014; 74: 2377-2384Crossref PubMed Scopus (250) Google Scholar). In order to select suitable in vitro models, the characterization of molecular features and their comparison to tumor tissues are needed. A detailed Cancer Cell Line Encyclopedia was recently established containing a genomic dataset for 947 human cancer cell lines, from which 58 are colorectal cancer lineages (17.Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. Wilson C.J. Lehár J. Kryukov G.V. Sonkin D. Reddy A. Liu M. Murray L. Berger M.F. Monahan J.E. Morais P. Meltzer J. Korejwa A. Jané-Valbuena J. Mapa F.A. Thibault J. Bric-Furlong E. Raman P. Shipway A. Engels I.H. Cheng J. Yu G.K. Yu J. Aspesi Jr., P. de Silva M. Jagtap K. Jones M.D. Wang L. Hatton C. Palescandolo E. Gupta S. Mahan S. Sougnez C. Onofrio R.C. Liefeld T. MacConaill L. Winckler W. Reich M. Li N. Mesirov J.P. Gabriel S.B. Getz G. Ardlie K. Chan V. Myer V.E. Weber B.L. Porter J. Warmuth M. Finan P. Harris J.L. Meyerson M. Golub T.R. Morrissey M.P. Sellers W.R. Schlegel R. Garraway L.A. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4822) Google Scholar). The Cancer Cell Line Encyclopedia includes data collections on genomic characterization, point mutation frequencies, DNA copy number, and mRNA expression levels. Comparison of these features between cell lines and primary tumors showed a high correlation in most cancer types, especially for colorectal cancer, suggesting that cell lines do represent tumor tissues quite reasonably at least on the genetic level. However, the number of publications characterizing cancer cell lines at a molecular level is far behind the number of articles using cancer cell lines as model systems (18.Ferreira D. Adega F. Chaves R. The importance of cancer cell lines as in vitro models in cancer methylome analysis and anticancer drugs testing oncogenomics and cancer proteomics - Novel Approaches in Biomarkers Discovery and Therapeutic Targets in Cancer, Dr. Cesar Lopez (Ed.).InTech. 2013; (DOI: 10.5772/53110)Google Scholar), and only few studies have been conducted on whether in vitro cultured cell lines can serve as suitable models for human tumors (19.Domcke S. Sinha R. Levine D.A. Sander C. Schultz N. Evaluating cell lines as tumour models by comparison of genomic profiles.Nat. Commun. 2013; 4: 2126Crossref PubMed Scopus (889) Google Scholar, 20.Sandberg R. Ernberg I. Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2052-2057Crossref PubMed Scopus (127) Google Scholar, 21.Ertel A. Verghese A. Byers S.W. Ochs M. Tozeren A. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells.Mol. Cancer. 2006; 5: 55Crossref PubMed Scopus (172) Google Scholar, 22.Chik J.H. Zhou J. Moh E.S. Christopherson R. Clarke S.J. Molloy M.P. Packer N.H. Comprehensive glycomics comparison between colon cancer cell cultures and tumours: Implications for biomarker studies.J. Proteomics. 2014; 108: 146-162Crossref PubMed Scopus (48) Google Scholar). Furthermore, cell lines are well characterized genetically, but they are largely understudied with regard to their glycosylation profiles. Here, we developed and optimized a new analytical method for the more sensitive and higher throughput N-glycome profiling of cells. This method is based on the release of N-glycans in a 96-well plate format from a PVDF-membrane (23.Burnina I. Hoyt E. Lynaugh H. Li H. Gong B. A cost-effective plate-based sample preparation for antibody N-glycan analysis.J. Chromatogr. A. 2013; 1307: 201-206Crossref PubMed Scopus (30) Google Scholar) starting from a low number of cells (250,000 cells), the chemical derivatization of released N-glycans enabling the stabilization and discrimination of α2,3- and α2,6-linked N-acetylneuraminic acids (24.Reiding K.R. Blank D. Kuijper D.M. Deelder A.M. Wuhrer M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.Anal. Chem. 2014; 86: 5784-5793Crossref PubMed Scopus (253) Google Scholar), followed by registration of the N-glycans by MALDI-TOF(/TOF)-MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems. Ammonium formiate, 2-aminobenzoic acid (AA), 2-picoline borane, dimethylsulfoxid (DMSO), 8 m guanidine hydrochloride (GuHCl), 1-hydroxybenzotriazole hydrate (HOBt), 50% sodium hydroxide, super DHB matrix (2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid, 1:9), and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO). HPLC SupraGradient acetonitrile (ACN) was obtained from Biosolve (Valkenswaard, The Netherlands). Dithiothreitol (DTT), ethanol, sodium bicarbonate (NaHCO3), and glacial acetic acid were from Merck (Darmstadt, Germany) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) from Fluorochem (Hadfield, UK). Peptide N-glycosidase F (PNGase F) was purchased from Roche Diagnostics (Mannheim, Germany). Recombinant PNGase F from Flavobacterium meningosepticum was a kind gift from Rick R. Drake (Medical University of South Carolina, SC) and Anand S. Mehta (Drexel University College of Medicine, PA). It was expressed and purified as described previously (25.Powers T.W. Jones E.E. Betesh L.R. Romano P.R. Gao P. Copland J.A. Mehta A.S. Drake R.R. Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.Anal. Chem. 2013; 85: 9799-9806Crossref PubMed Scopus (122) Google Scholar) and is commercially available from Bulldog Bio (Portsmouth, NH) as PNGase F Prime™. The peptide calibration standard was purchased from Bruker Daltonics (Bremen, Germany). MultiScreen® HTS 96 multiwell plates (pore size 0.45 μm) with high protein-binding membrane (hydrophobic Immobilon-P PVDF membrane) were purchased from Millipore (Amsterdam, The Netherlands), conical 96-well Nunc plates from Thermo Scientific (Roskilde, Denmark). Hepes-buffered RPMI 1640 and Dulbecco's Modified Eagle (DMEM) culture media were purchased from Gibco (Paisley, UK), and l-glutamine from Gibco and Lonza (Basel, Switzerland), penicillin and streptomycin from MP Biomedicals (Santa Ana, CA), Lonza, 10x trypsin/EDTA solution (5.0 g/l porcine trypsin and 2.0 g/l EDTA●4Na in 0.9% sodium chloride) from PAA Laboratories GmbH (Pasching, Austria), fetal calf serum (FCS) from PAA Laboratories and Biowest (Alkmaar, The Netherlands), and T75 cell culture flasks from Greiner-Bio One B.V. (Alphen a/d Rijn, The Netherlands). All buffers were prepared using Milli-Q water (mQ) generated from a Q-Gard 2 system (Millipore). Control Visucon-F plasma pool (citrated and 0.02 m Hepes buffered plasma pool from 20 healthy human donors) was obtained from Affinity Biologicals (Ancaster, Canada). The mRNA Capture kit was obtained from Roche and the reverse transcription system kit from Promega (Madison, WI). Human colorectal cancer cell lines (see Table I and Supplemental Table S1) were obtained from the Department of Surgery of the Leiden University Medical Center (LUMC), Leiden, The Netherlands, as well as the Department of Pathology of the VU University Medical Center (VUmc), Amsterdam, The Netherlands. Cells were cultured in Hepes-buffered RPMI 1640 culture medium containing l-glutamine and supplemented with penicillin (5000 IU/ml), streptomycin (5 mg/ml), and 10% (v/v) fetal calf serum (FCS) at the LUMC or in DMEM medium, supplemented with 10% (v/v) FCS and antibiotics, except for the KM12 cell line, which was grown in RPMI/10% FCS/antibiotics and l-glutamine at the VUmc. Cells were incubated at 37 °C with 5% CO2 in humidified air and cell culturing was performed up to a confluence of 80% under sterile conditions. For harvesting of the cells, medium was removed and adherent cells were washed twice with 1x PBS and trypsinized using 1x trypsin/EDTA solution in 1x PBS. To stop trypsin activity, medium in a ratio of 2:5 (trypsin/EDTA/medium; v/v) was added and cells were pelleted at 300 × g for 5 min. Cells were then resuspended in 3 ml 1x PBS and counted using the CountessTM Automated Cell Counter (Invitrogen, Paisley, UK) based on tryptan blue staining. Cells were aliquoted to 2.0 × 106 cells per ml of 1x PBS, and washed twice with 1 ml 1x PBS for 3 min at 1000 × g. The supernatant was removed and pellets stored at −20 °C until further use.Table IOverview of colorectal cancer cell linesNo.SampleStagingTumor siteGenderAge1C102PrimaryMale712CaCo22PrimaryMale723Co1153PrimaryFemale774Colo205_VUmc4Ascitic fluid (metastatic site)bTreated with 5-fluorouracil for 4–6 weeks before collection.Male705Colo320_VUmc3PrimaryFemale586DLD-1aDNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile.3PrimaryMale457HCT1164PrimaryMale487bHCT116_VUmc4PrimaryMale488HCT15_VUmcaDNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile.3PrimaryMale9HCT8aDNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile.3—Male6710HT29cDerivatives.3PrimaryFemale4410bHT29_VUmc3PrimaryFemale4411KM12_VUmc2Primary——12LOVO3Lymph node metastasisMale5613LS174T_VUmcdVariants/sister cell lines.2PrimaryFemale5814LS-180dVariants/sister cell lines.2PrimaryFemale5815LS-411N2PrimaryMale3216RKO_VUmc————17SW11161PrimaryMale7318SW1398_VUmc————19SW14633PrimaryFemale6620SW483PrimaryFemale8221SW480eSame patient.2PrimaryMale5121bSW480_VUmceSame patient.2PrimaryMale5122SW620eSame patient.3Lymph node metastasisMale5123SW948_VUmc3PrimaryFemale8124T84—Lung metastasisMale7225WiDrcDerivatives.—PrimaryFemale78a DNA profiling studies (57, 58) have shown that DLD-1, HCT-15, HCT-8 and HRT-18G share a single profile.b Treated with 5-fluorouracil for 4–6 weeks before collection.c Derivatives.d Variants/sister cell lines.e Same patient. Open table in a new tab Cell pellets (∼2 × 106 cells) from two or three biological replicates of each colorectal cancer cell line (see Table I and Supplemental Table S1) were suspended in 100 μl mQ and sonicated in a water bath for 30 min. As control, 20 μl of human control Visucon-F plasma was used that was brought to 100 μl with mQ. Glycans were released using a PVDF-membrane based protocol adapted from Burnina et al. (23.Burnina I. Hoyt E. Lynaugh H. Li H. Gong B. A cost-effective plate-based sample preparation for antibody N-glycan analysis.J. Chromatogr. A. 2013; 1307: 201-206Crossref PubMed Scopus (30) Google Scholar). Shortly, 0.25 × 106 cells/well or 25 μl/well diluted human plasma in denaturation buffer (5.8 m GuHCl and 5 mm DTT), were loaded in quadruplicate onto preconditioned HTS 96-well plates with hydrophobic Immobilon-P PVDF membrane and incubated for 30 min at 60 °C in a moistured, sealed box as an incubation chamber within an oven. Plates were shaken for 5 min on a horizontal shaker prior to centrifugation (1 min, 500 × g). The wells were washed twice with 200 μl mQ with 2 min incubation steps on a horizontal shaker prior to centrifugation and once with 200 μl 100 mm NaHCO3 (1 min, 500 × g). For N-glycan release, 50 μl 100 mm NaHCO3 and 1 mU PNGase F (Roche) were added per well. Plates were placed into the incubation device and incubated for 3 h at 37 °C. Glycans were recovered into 96-well collection plates by centrifugation (2 min, 1000 × g); eventual residual solution was collected from the membrane. A subset of CRC cell lines were used to obtain the cell surface N-glycome in comparison to the N-glycosylation profile of the residual pellet using a protocol modified from Hamouda et al. (26.Hamouda H. Kaup M. Ullah M. Berger M. Sandig V. Tauber R. Blanchard V. Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry.J. Proteome Res. 2014; 13: 6144-6151Crossref PubMed Scopus (36) Google Scholar). CRC cells were cultured as described above. Cells were harvested, pelleted, and aliquoted in Eppendorf tubes to 4 × 106 cells in 1x PBS. Subsequently, cells were washed 2 × 10 min and 1 × 5 min at 300 × g with 500 μl 1x PBS. Next, cell pellets were carefully dissolved in 500 μl 1x PBS and 3.5 μl recombinant PNGase F (∼9 μg) were added and samples incubated for 30 min at 37 °C and 250 rpm in an Innova 43 incubator shaker (New Brunswick, Enfield, CT). Afterwards, samples were centrifuged 15 min at 500 × g and the supernatant containing the released glycans was collected. Supernatant and residual pellet were stored separately at −20 °C until further use. N-glycans from residual pellets were then released using the PVDF-membrane based protocol as described, but with 1 million cells per well and overnight PNGase F incubation. Released N-glycans were derivatized by ethyl esterification adapted from Reiding et al. (24.Reiding K.R. Blank D. Kuijper D.M. Deelder A.M. Wuhrer M. High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.Anal. Chem. 2014; 86: 5784-5793Crossref PubMed Scopus (253) Google Scholar) allowing for discrimination of N-acetylneuraminic acid linkages (α2,3 versus α2,6). Briefly, 20 μl of released N-glycans from the total and residual cell pellets as well as the control samples were added to 100 μl of ethyl esterification reagent (0.25 m EDC and 0.25 m HOBt, 1:1 v/v). For the cell surface glycans, the supernatant was concentrated in a vacuum centrifuge to a volume of 20–30 μl, and 100 μl ethyl esterification reagent was added. Samples were incubated for 1 h at 37 °C. Subsequently, 100 μl ACN were added and the mixture was incubated at −20 °C for 15 min. Samples were brought to room temperature prior to glycan purification by hydrophilic interaction liquid chromatography (HILIC) solid phase extraction modified from a protocol described previously (27.Selman M.H. Hemayatkar M. Deelder A.M. Wuhrer M. Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides.Anal. Chem. 2011; 83: 2492-2499Crossref PubMed Scopus (251) Google Scholar). For purification of pellet derived N-glycans, pipette tips of 20 μl volume were packed with a 4 mm piece of cotton thread (ca. 200 μg of cotton), equilibrated by pipetting 3 × 20 μl mQ water, followed by 3 × 20 μl 85% ACN. Samples were loaded by carefully pipetting up and down for 50 times. The tips were washed by pipetting 3 × 20 μl 85% ACN/1% TFA and 3 × 20 μl 85% ACN. N-glycans were eluted in 10 μl mQ water. For cell surface glycans, pipette tips of 200 μl volume were packed with cotton wool (ca 1000 μg) to prevent clogging through salts. The cotton HILIC purification was performed as described but with 150 μl pipetting volume, while elution was kept at 10 μl mQ water. For mass spectrometric analysis, 5 μl of derivatized N-glycans were spotted onto an anchor chip MALDI target plate (Bruker Daltonics) and cocrystallized with 1 μl of 1 mg/ml superDHB in ACN/mQ (1/1, v/v) containing 1 mm NaOH. Samples were allowed to dry at room temperature and were then recrystallized with 0.5 μl 5 mg/ml superDHB in ACN/mQ (1/1, v/v) containing 1 mm NaOH. MALDI-TOF-MS spectra were acquired using an UltrafleXtremeTM mass spectrometer in the positive-ion reflector mode, controlled by FlexControl 3.4 software Build 119 (Bruker Daltonics). The instrument was calibrated using a Bruker peptide calibration kit. Spectra were obtained over a mass window of m/z 1000–5000 with ion suppression below m/z 900 for a total of 10,000 shots (1000 Hz laser frequency, 200 shots per raster spot during complete random walk). Tandem mass spectrometry (MALDI-TOF-MS/MS) was performed for structural elucidation via fragmentation in gas-off TOF/TOF mode. A mean average spectrum over all the total cell line sample spectra was generated using an in-house developed script in Python 2.7.3 (Python Software Foundation; http://docs.python.org/py3k/reference/index.html). The average spectrum was internally recalibrated using glycan peaks of known composition (Supplemental Table S2), smoothed (Savitzky Golay algorithm, peak width: m/z 0.06, four cycles), and baseline-corrected (Tophat algorithm) using FlexAnalysis Software (Version 3.3; Bruker Daltonics). Peaks of signal-to-noise > 2 were picked, manually revised, and analyzed in GlycoWorkbench 2.1 stable build 146 (http://www.eurocarbdb.org/) using the Glyco-Peakfinder tool (http://www.eurocarbdb.org/ms-tools/) for generation of a glycan compositions list. Using MassyTools version 0.1.5.1, which is a novel in-house software developed for automated data processing, the resulting glycan peak list generated based on the average spectrum was used for targeted data extraction of the a

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