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Developments in the Identification of Glycan Biomarkers for the Detection of Cancer

2013; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês

10.1074/mcp.r112.026799

1535-9484

L. Renee Ruhaak, Suzanne Miyamoto, Carlito B. Lebrilla,

Carbohydrate Chemistry and Synthesis

Changes in glycosylation readily occur in cancer and other disease states. Thanks to recent advances in the development of analytical techniques and instrumentation, especially in mass spectrometry, it is now possible to identify blood-derived glycan-based biomarkers using glycomics strategies. This review is an overview of the developments made in the search for glycan-based cancer biomarkers and the technologies currently in use. It is anticipated that the progressing instrumental and bioinformatics developments will allow the identification of relevant glycan biomarkers for the diagnosis, early detection, and monitoring of cancer treatment with sufficient sensitivity and specificity for clinical use. Changes in glycosylation readily occur in cancer and other disease states. Thanks to recent advances in the development of analytical techniques and instrumentation, especially in mass spectrometry, it is now possible to identify blood-derived glycan-based biomarkers using glycomics strategies. This review is an overview of the developments made in the search for glycan-based cancer biomarkers and the technologies currently in use. It is anticipated that the progressing instrumental and bioinformatics developments will allow the identification of relevant glycan biomarkers for the diagnosis, early detection, and monitoring of cancer treatment with sufficient sensitivity and specificity for clinical use. The ubiquity of glycosylation and the fundamental importance of N-glycans in nearly every biological process give N-glycans high potential as biomarkers. Nearly 70% of the plasma proteome is glycosylated, and most cell membranes and secreted proteins are also highly glycosylated. The N-glycans play important roles in major biological processes that include cell–cell and cell–matrix interactions (1Gu J. Isaji T. Xu Q. Kariya Y. Gu W. Fukuda T. Du Y. Potential roles of N-glycosylation in cell adhesion.Glycoconj. J. 2012; 29: 599-607Crossref PubMed Scopus (114) Google Scholar), protein folding (2Shental-Bechor D. Levy Y. Folding of glycoproteins: toward understanding the biophysics of the glycosylation code.Curr. Opin. Struct. Biol. 2009; 19: 524-533Crossref PubMed Scopus (182) Google Scholar), receptor binding, and protein clearance (3Fukuda M.N. Sasaki H. Lopez L. Fukuda M. Survival of recombinant erythropoietin in the circulation: the role of carbohydrates.Blood. 1989; 73: 84-89Crossref PubMed Google Scholar). There is ample evidence that altered N-glycosylation patterns are present on tumor cells (e.g. Refs. 4Balog 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, 5Dube D.H. Bertozzi C.R. Glycans in cancer and inflammation—potential for therapeutics and diagnostics.Nat. Rev. Drug Discov. 2005; 4: 477-488Crossref PubMed Scopus (1349) Google Scholar, 6Mehta A. Norton P. Liang H. Comunale M.A. Wang M. Rodemich-Betesh L. Koszycki A. Noda K. Miyoshi E. Block T. Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue.Cancer Epidemiol. Biomarkers Prev. 2012; 21: 925-933Crossref PubMed Scopus (50) Google Scholar, 7de Leoz M.L. Young L.J. An H.J. Kronewitter S.R. Kim J. Miyamoto S. Borowsky A.D. Chew H.K. Lebrilla C.B. High-mannose glycans are elevated during breast cancer progression.Mol. Cell. Proteomics. 2011; 10M110.002717Abstract Full Text Full Text PDF PubMed Google Scholar), and these findings have sparked the search for glycan-based biomarkers for the detection of different types of cancer in biofluids such as serum and plasma (8Packer N.H. von der Lieth C.W. Aoki-Kinoshita K.F. Lebrilla C.B. Paulson J.C. Raman R. Rudd P. Sasisekharan R. Taniguchi N. York W.S. Frontiers in glycomics: bioinformatics and biomarkers in disease. An NIH white paper prepared from discussions by the focus groups at a workshop on the NIH campus, Bethesda MD (September 11–13, 2006).Proteomics. 2008; 8: 8-20Crossref PubMed Scopus (134) Google Scholar). Glycans are highly branched structures built up of monosaccharides that are linked by glycosidic bonds in multiple different ways in a non-template-driven manner. This is in contrast to proteins, which consist of amino acids linked by well-defined peptide bonds in a linear sequence-dependent template-driven fashion. It has been speculated that over 3000 N-glycan structures might exist based on the number of possible monosaccharides and the different linkages that might occur among them (9Goldberg D. Sutton-Smith M. Paulson J. Dell A. Automatic annotation of matrix-assisted laser desorption/ionization N-glycan spectra.Proteomics. 2005; 5: 865-875Crossref PubMed Scopus (144) Google Scholar). The number corresponds well to recent estimates based on a theoretical library of serum glycans in which the actual size of the human serum N-glycome is proposed to be restricted to around 300 to 500 compositions corresponding to fewer than 5000 structures (10Kronewitter S.R. An H.J. de Leoz M.L. Lebrilla C.B. Miyamoto S. Leiserowitz G.S. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.Proteomics. 2009; 9: 2986-2994Crossref PubMed Scopus (105) Google Scholar). The pathways involved in N-glycan biosynthesis are generally well understood and have been reviewed extensively (11Kornfeld R. Kornfeld S. Assembly of asparagine-linked oligosaccharides.Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3772) Google Scholar). Briefly, a dolichol precursor is enzymatically decorated with a glycan precursor (GlcNAc2Man9Glc3), and the dolichol-glycan precursor in the endoplasmic reticulum is then transferred to the target protein. When the protein is folded correctly and transferred to the Golgi, the glycan precursor is modified from a high-mannose structure to the mature N-glycan using several glycosyltransferases and glycosidases. Although this process is well regulated, individual proteins might contain more than one glycosylation site and are often decorated with different glycans on the different sites. Moreover, multiple copies of the same protein are likely to have different glycans attached, leading to large glycan microheterogeneities, further complicating glycan analysis. Over the years, several approaches have been used for the analysis of glycoproteins and N-glycans. Early studies mostly evaluated lectin binding, showing the presence of certain binding epitopes on specific proteins, cells, or tissues. More structurally extensive methods, including HPLC and, especially, mass spectrometry, have expanded the field of glycomics (12Wuhrer M. Glycomics using mass spectrometry.Glycoconj. J. 2013; 30: 11-22Crossref PubMed Scopus (120) Google Scholar). Several analytical approaches have been used for the profiling of N-glycans, including stand-alone mass spectrometry and separation strategies such as reverse phase chromatography, hydrophilic interaction chromatography (HILIC) (13Wuhrer M. de Boer A.R. Deelder A.M. Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry.Mass Spectrom. Rev. 2009; 28: 192-206Crossref PubMed Scopus (205) Google Scholar, 14Zauner G. Deelder A.M. Wuhrer M. Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics.Electrophoresis. 2011; 32: 3456-3466Crossref PubMed Scopus (147) Google Scholar), porous graphitic carbon (PGC) (15Ruhaak L.R. Deelder A.M. Wuhrer M. Oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry.Anal. Bioanal. Chem. 2009; 394: 163-174Crossref PubMed Scopus (151) Google Scholar) chromatography, and capillary electrophoresis (16Mechref Y. Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry.Electrophoresis. 2011; 32: 3467-3481Crossref PubMed Scopus (51) Google Scholar), which have all been reviewed extensively. Currently, a comprehensive glycomics approach that yields structures as well as quantitative information involves the profiling of serum or plasma glycans by means of mass spectrometry. Because diseases such as cancer alter glycosylation, a systems-wide analysis of the glycome could yield direct changes in health conditions even without consideration of protein identification or abundances. Indeed, a glycomic analysis of serum proteins indicated that many proteins change their glycosylation simultaneously in disease states relative to the control (17Li B. An H.J. Kirmiz C. Lebrilla C.B. Lam K.S. Miyamoto S. Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins.J. Proteome Res. 2008; 7: 3776-3788Crossref PubMed Scopus (60) Google Scholar, 18Dempsey E. Rudd P.M. Acute phase glycoproteins: bystanders or participants in carcinogenesis?.Ann. N. Y. Acad. Sci. 2012; 1253: 122-132Crossref PubMed Scopus (33) Google Scholar). The first global glycomics approach investigating serum was performed with serum from ovarian cancer patients (19An H.J. Miyamoto S. Lancaster K.S. Kirmiz C. Li B. Lam K.S. Leiserowitz G.S. Lebrilla C.B. Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer.J. Proteome Res. 2006; 5: 1626-1635Crossref PubMed Scopus (190) Google Scholar). The intrinsic complexity of glycosylation allows the discovery of biomarkers at various levels of structural analysis. The first types of biomarkers were based solely on compositions (the monosaccharides present in a specific glycan), which can be accurately monitored via stand-alone mass spectrometry (7de Leoz M.L. Young L.J. An H.J. Kronewitter S.R. Kim J. Miyamoto S. Borowsky A.D. Chew H.K. Lebrilla C.B. High-mannose glycans are elevated during breast cancer progression.Mol. Cell. Proteomics. 2011; 10M110.002717Abstract Full Text Full Text PDF PubMed Google Scholar, 20Kronewitter S.R. De Leoz M.L. Strum J.S. An H.J. Dimapasoc L.M. Guerrero A. Miyamoto S. Lebrilla C.B. Leiserowitz G.S. The glycolyzer: automated glycan annotation software for high performance mass spectrometry and its application to ovarian cancer glycan biomarker discovery.Proteomics. 2012; 12: 2523-2538Crossref PubMed Scopus (43) Google Scholar). Compositional analysis can yield the numbers of hexoses, N-acetylhexoses, fucoses, and sialic acids. Variations in the number of antennae, the number of terminal galactoses, and the degree of sialylation and fucosylation can all be inferred solely from mass spectral data (e.g. Refs. 21Ruhaak L.R. Huhn C. Waterreus W.J. de Boer A.R. Neususs C. Hokke C.H. Deelder A.M. Wuhrer M. Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins.Anal. Chem. 2008; 80: 6119-6126Crossref PubMed Scopus (177) Google Scholar, 22Harvey D.J. Structural determination of N-linked glycans by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.Proteomics. 2005; 5: 1774-1786Crossref PubMed Scopus (120) Google Scholar, 23Guillard M. Gloerich J. Wessels H.J. Morava E. Wevers R.A. Lefeber D.J. Automated measurement of permethylated serum N-glycans by MALDI-linear ion trap mass spectrometry.Carbohydr. Res. 2009; 344: 1550-1557Crossref PubMed Scopus (30) Google Scholar, 24Stumpo K.A. Reinhold V.N. The N-glycome of human plasma.J. Proteome Res. 2010; 9: 4823-4830Crossref PubMed Scopus (70) Google Scholar). A combination of techniques allowing isomer separation with mass spectrometry improves the analysis. Each N-glycan composition can yield a number of isomers that have the same composition but different arrangements of monosaccharide linkages. Nanoflow liquid chromatography (nLC) employing PGC stationary phase coupled with mass spectrometry allows the effective separation of native glycan structures with relative and potentially absolute quantitation (25Chu C.S. Ninonuevo M.R. Clowers B.H. Perkins P.D. An H.J. Yin H. Killeen K. Miyamoto S. Grimm R. Lebrilla C.B. Profile of native N-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry.Proteomics. 2009; 9: 1939-1951Crossref PubMed Scopus (128) Google Scholar, 26Hua S. An H.J. Ozcan S. Ro G.S. Soares S. DeVere-White R. Lebrilla C.B. Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers.Analyst. 2011; 136: 3663-3671Crossref PubMed Scopus (125) Google Scholar, 27Hua S. Lebrilla C. An H.J. Application of nano-LC-based glycomics towards biomarker discovery.Bioanalysis. 2011; 3: 2573-2585Crossref PubMed Scopus (53) Google Scholar, 28Ruhaak L.R. Miyamoto S. Kelly K. Lebrilla C.B. N-glycan profiling of dried blood spots.Anal. Chem. 2012; 84: 396-402Crossref PubMed Scopus (61) Google Scholar). Using this method, biomarkers can now be discovered on a compound specific level. The potentially most specific, but also technically most difficult, are protein-specific and site-specific glycosylation. The analytical approach employs tools for protein-specific analysis by isolating proteins for glycan analysis and possibly specific glycopeptides. Because of the technical difficulty of these methods, they have been applied primarily to a very small group of highly abundant glycoproteins, namely, IgG. The glycan compositions carried by individual proteins are characterized using protein digestion by either specific or nonspecific proteases and subsequent analysis of the glycopeptides (29Clowers B.H. Dodds E.D. Seipert R.R. Lebrilla C.B. Site determination of protein glycosylation based on digestion with immobilized nonspecific proteases and Fourier transform ion cyclotron resonance mass spectrometry.J. Proteome Res. 2007; 6: 4032-4040Crossref PubMed Scopus (62) Google Scholar). This strategy, combined with data-interpretation software, is used to identify site-specific glycosylation patterns (30Nwosu C.C. Seipert R.R. Strum J.S. Hua S.S. An H.J. Zivkovic A.M. German B.J. Lebrilla C.B. Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.J. Proteome Res. 2011; 10: 2612-2624Crossref PubMed Scopus (108) Google Scholar, 31Hua S. Nwosu C.C. Strum J.S. Seipert R.R. An H.J. Zivkovic A.M. German J.B. Lebrilla C.B. Site-specific protein glycosylation analysis with glycan isomer differentiation.Anal. Bioanal. Chem. 2012; 403: 1291-1302Crossref PubMed Scopus (90) Google Scholar). Although it is highly desirable to obtain site-specific information at the individual protein level of glycan biomarkers, the complexity of such analyses does not yet allow the profiling of complex mixtures of proteins. Therefore, this review focuses on the first two analytical strategies, utilizing MALDI coupled with Fourier transform ion cyclotron resonance (FTICR) MS and nLC-PGC-TOF-MS, and their use in biomarker discovery. The structural schemes of glycans are dependent on their compositions, which in humans consist primarily of hexoses, N-acetyl hexosamines, fucoses, and N-acetyl neuraminic acids. Each individual N-glycan composition has a specific mass, and therefore direct mass spectrometric techniques such as MALDI-MS and direct infusion electrospray ionization MS are ideal tools for the compositional analysis of N-glycans. There are two major approaches to glycan mass or compositional profiling used in biomarker discovery, which are based on whether to perderivatize or leave the glycans in their native state for MS analysis. Permethylation is used because it increases the sensitivity to MS analysis and stabilizes labile groups such as sialic acids, particularly during MALDI ionization, which is more energetic than electrospray ionization. However, permethylation is a chemical process, and exposing the glycans to chemical reactions could result in degradation, such as the loss of sialic acids. Furthermore, partially methylated products can be generated, producing a background of chemical noise that could effectively decrease the overall dynamic range of the method. The choice is probably dictated by the comfort level of the laboratory with one method or the other, as both have been shown to generally yield the same numbers of glycan peaks in the mass spectra. A method developed in our laboratory illustrates the analysis of native glycans. It is a high-throughput glycomics workflow for serum and plasma using MALDI and high-performance ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) (32de Leoz M.L. An H.J. Kronewitter S. Kim J. Beecroft S. Vinall R. Miyamoto S. de Vere W.R. Lam K.S. Lebrilla C. Glycomic approach for potential biomarkers on prostate cancer: profiling of N-linked glycans in human sera and pRNS cell lines.Dis. Markers. 2008; 25: 243-258Crossref PubMed Scopus (74) Google Scholar, 33Kronewitter S.R. de Leoz M.L. Peacock K.S. McBride K.R. An H.J. Miyamoto S. Leiserowitz G.S. Lebrilla C.B. Human serum processing and analysis methods for rapid and reproducible N-glycan mass profiling.J. Proteome Res. 2010; 9: 4952-4959Crossref PubMed Scopus (63) Google Scholar). The method employs the enzymatic release of N-glycans using PNGaseF; other chemical glycan-release methods have been developed and are reviewed elsewhere (e.g. 34Ruhaak L.R. Zauner G. Huhn C. Bruggink C. Deelder A.M. Wuhrer M. Glycan labeling strategies and their use in identification and quantification.Anal. Bioanal. Chem. 2010; 397: 3457-3481Crossref PubMed Scopus (354) Google Scholar). To speed up the glycan-release procedure, it is performed in a microwave reactor, which reduces the time needed for glycan release from 16 h or overnight to 10 min. An alternative method that could be used to shorten the N-glycan release time might be pressure-cycling technology (35Szabo Z. Guttman A. Karger B.L. Rapid release of N-linked glycans from glycoproteins by pressure-cycling technology.Anal. Chem. 2010; 82: 2588-2593Crossref PubMed Scopus (61) Google Scholar). Upon release, the N-glycans are purified using a cartridge containing PGC in which the glycans are eluted in three fractions (32de Leoz M.L. An H.J. Kronewitter S. Kim J. Beecroft S. Vinall R. Miyamoto S. de Vere W.R. Lam K.S. Lebrilla C. Glycomic approach for potential biomarkers on prostate cancer: profiling of N-linked glycans in human sera and pRNS cell lines.Dis. Markers. 2008; 25: 243-258Crossref PubMed Scopus (74) Google Scholar) of varying proportions of acetonitrile in water to yield (i) a fraction containing neutral glycans, with enrichment of high-mannose-type glycans, (ii) mostly neutral hybrid and complex type glycans, and (iii) mostly hybrid and complex type glycans containing N-acetyl neuraminic acid residues (Fig. 1). The high accuracy of the FTICR allows the unambiguous identification of glycan compositions based on the accurate mass in combination with a retrosynthetic glycan composition library (10Kronewitter S.R. An H.J. de Leoz M.L. Lebrilla C.B. Miyamoto S. Leiserowitz G.S. The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.Proteomics. 2009; 9: 2986-2994Crossref PubMed Scopus (105) Google Scholar). Using the strategy described, 64 glycan compositions can be detected consistently from serum with an average coefficient of variation (cv) of less than 10% (33Kronewitter S.R. de Leoz M.L. Peacock K.S. McBride K.R. An H.J. Miyamoto S. Leiserowitz G.S. Lebrilla C.B. Human serum processing and analysis methods for rapid and reproducible N-glycan mass profiling.J. Proteome Res. 2010; 9: 4952-4959Crossref PubMed Scopus (63) Google Scholar). The application of this method to a smaller set of sera from mice transplanted with human tumor tissue and sera from breast cancer patients compared with that of healthy controls showed increased levels of high-mannose-type structures with cancer in both mouse and human sera (7de Leoz M.L. Young L.J. An H.J. Kronewitter S.R. Kim J. Miyamoto S. Borowsky A.D. Chew H.K. Lebrilla C.B. High-mannose glycans are elevated during breast cancer progression.Mol. Cell. Proteomics. 2011; 10M110.002717Abstract Full Text Full Text PDF PubMed Google Scholar). A second sample set containing serum from ovarian cancer patients compared with serum from matched controls recently revealed increased levels of sialylated glycans, whereas several neutral glycans, including high-mannose-type glycans, were decreased. Importantly, levels of a small group of truncated glycans (Hex3HexNAc4, Hex3HexNAc4Fuc1, Hex3HexNAc5, and Hex3HexNAc5Fuc1) were increased (20Kronewitter S.R. De Leoz M.L. Strum J.S. An H.J. Dimapasoc L.M. Guerrero A. Miyamoto S. Lebrilla C.B. Leiserowitz G.S. The glycolyzer: automated glycan annotation software for high performance mass spectrometry and its application to ovarian cancer glycan biomarker discovery.Proteomics. 2012; 12: 2523-2538Crossref PubMed Scopus (43) Google Scholar). A similar approach has been used by Kim et al. (36Kim Y.G. Jeong H.J. Jang K.S. Yang Y.H. Song Y.S. Chung J. Kim B.G. Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform.Anal. Biochem. 2009; 391: 151-153Crossref PubMed Scopus (28) Google Scholar), who applied MALDI-TOF-MS for the detection of biomarkers for ovarian cancer in desialylated serum samples and found increased levels of the fucosylated bi- and tri-antennary glycans Hex3HexNAc4Fuc1 and Hex6HexNAc5Fuc1. The permethylation of glycans before analysis eliminates the need for fractionation as performed in the analysis of native glycans and allows the whole mixture to be examined simultaneously, typically with MALDI-TOF-MS (37Haslam S.M. North S.J. Dell A. Mass spectrometric analysis of N- and O-glycosylation of tissues and cells.Curr. Opin. Struct. Biol. 2006; 16: 584-591Crossref PubMed Scopus (95) Google Scholar). Although permethylation is a relatively harsh chemical reaction, its products, and especially the N-glycans containing sialic acids, are known to be more stable in the MALDI ionization process. Because oligosaccharides containing sialic acid are the most abundant species in serum, these studies tend to focus more on sialylated components. The application of this method to a set of breast cancer samples revealed a significant increase in fucosylated and sialylated glycans (38Kyselova Z. Mechref Y. Kang P. Goetz J.A. Dobrolecki L.E. Sledge G.W. Schnaper L. Hickey R.J. Malkas L.H. Novotny M.V. Breast cancer diagnosis and prognosis through quantitative measurements of serum glycan profiles.Clin. Chem. 2008; 54: 1166-1175Crossref PubMed Scopus (204) Google Scholar). Moreover, in a separate study, three glycans were shown to provide good sensitivity and specificity for the separation of serum samples from patients with hepatocellular carcinoma and controls (39Goldman R. Ressom H.W. Varghese R.S. Goldman L. Bascug G. Loffredo C.A. Abdel-Hamid M. Gouda I. Ezzat S. Kyselova Z. Mechref Y. Novotny M.V. Detection of hepatocellular carcinoma using glycomic analysis.Clin. Cancer Res. 2009; 15: 1808-1813Crossref PubMed Scopus (121) Google Scholar). Application of the method to a set of ovarian cancer samples showed that the levels of tri- and tetra-antennary N-glycans were increased, independent of their levels of fucosylation and sialylation (40Alley Jr., W.R. Vasseur J.A. Goetz J.A. Svoboda M. Mann B.F. Matei D.E. Menning N. Hussein A. Mechref Y. Novotny M.V. N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients.J. Proteome Res. 2012; 11: 2282-2300Crossref PubMed Scopus (153) Google Scholar). Moreover, levels of glycans containing a bisecting GlcNAc were shown to be decreased (40Alley Jr., W.R. Vasseur J.A. Goetz J.A. Svoboda M. Mann B.F. Matei D.E. Menning N. Hussein A. Mechref Y. Novotny M.V. N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients.J. Proteome Res. 2012; 11: 2282-2300Crossref PubMed Scopus (153) Google Scholar). Separation methods including LC or capillary electrophoresis with fluorescence or UV detection are useful once structures have been completely or partially elucidated. The most widely applied technique for separation is HILIC-HPLC of 2-AB-labeled 1The abbreviations used are:2-AB2-aminobenzamideFTICRFourier transform ion cyclotron resonanceHILIChydrophilic interaction chromatographyMALDImatrix-assisted laser desorption ionizationnLCnanoflow liquid chromatographyPGCporous graphitic carbonTOFtime of flightUPLCultra performance liquid chromatography. glycans (41Royle L. Campbell M.P. Radcliffe C.M. White D.M. Harvey D.J. Abrahams J.L. Kim Y.G. Henry G.W. Shadick N.A. Weinblatt M.E. Lee D.M. Rudd P.M. Dwek R.A. HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software.Anal. Biochem. 2008; 376: 1-12Crossref PubMed Scopus (385) Google Scholar, 42Bones J. Mittermayr S. O'Donoghue N. Guttman A. Rudd P.M. Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.Anal. Chem. 2010; 82: 10208-10215Crossref PubMed Scopus (155) Google Scholar), which separates oligosaccharides mostly by their size. Using a glucose index, retention times can be matched and glycan structures may be assigned. The resolution of HILIC-HPLC is relatively poor, but increased resolution may be obtained using ultra performance liquid chromatography (UPLC), with ∼17 (HPLC) and 45 (UPLC) respective peaks typically observed in serum samples when using these techniques. Although the use of fluorescence allows accurate and robust quantitation, the incidence of overlapping glycans usually requires the use of exoglycosidases for unambiguous identification of the candidate biomarkers. 2-aminobenzamide Fourier transform ion cyclotron resonance hydrophilic interaction chromatography matrix-assisted laser desorption ionization nanoflow liquid chromatography porous graphitic carbon time of flight ultra performance liquid chromatography. HILIC-HPLC with fluorescence detection has been widely applied for the identification of candidate biomarkers for a wide range of cancer types, including gastric, lung, ovarian, and breast cancer and those results have been reviewed recently (43Adamczyk B. Tharmalingam T. Rudd P.M. Glycans as cancer biomarkers.Biochim. Biophys. Acta. 2012; 1820: 1347-1353Crossref PubMed Scopus (382) Google Scholar). It was observed that in patients suffering from gastric cancer, levels of fucosylated non- and mono-sialylated glycans were decreased, whereas a peak containing disialylated biantennary glycan (Hex5HexNAc4Sia2) showed increased levels (42Bones J. Mittermayr S. O'Donoghue N. Guttman A. Rudd P.M. Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.Anal. Chem. 2010; 82: 10208-10215Crossref PubMed Scopus (155) Google Scholar). In a second study, two peaks, one containing biantennary asialo monogalactosylated glycans and one containing triantennary glycans carrying α2–3-linked sialic acids, were decreased with gastric cancer, and one peak containing triantennary glycans carrying α2–6-linked sialic acids and trisialylated triantennary glycans carrying sialyl Lewis x showed increased levels (44Bones J. Byrne J.C. O'Donoghue N. McManus C. Scaife C. Boissin H. Nastase A. Rudd P.M. Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms.J. Proteome Res. 2011; 10: 1246-1265Crossref PubMed Scopus (112) Google Scholar). In a study of serum samples from lung cancer patients and controls, peaks containing mostly tri- and tetra-antennary, highly sialylated glycans, some with antenna and some with core fucosylation, were increased. A significant decrease was observed for peaks containing mostly biantennary glycans, mostly with core fucose (45Arnold J.N. Saldova R. Galligan M.C. Murphy T.B. Mimura-Kimura Y. Telford J.E. Godwin A.K. Rudd P.M. Novel glycan biomarkers for the detection of lung cancer.J. Proteome Res. 2011; 10: 1755-1764Crossref PubMed Scopus (162) Google Scholar). When serum profiles of ovarian cancer patients are compared with controls, the most profound changes include the increased levels of peaks containing core fucosylated agalactosyl biantennary glycans and peaks that consist of sialyl-Lewis-x-containing glycans (46Saldova R. Royle L. Radcliffe C.M. Abd Hamid U.M. Evans R. Arnold J.N. Banks R.E. Hutson R. Harvey D.J. Antrobus R. Petrescu S.M. Dwek R.A. Rudd P.M. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.Glycobiology. 2007; 17: 1344-1356Crossref PubMed Scopus (330) Google Scholar). In a separate study, significantly increased levels of peaks containing highly sialylated antenna fucosylated glycans (carrying sialyl Lewis x) were also observed in serum samples from advanced-stage breast cancer patients relative to controls (47Saldova R. Reuben J.M. Abd Hamid U.M. Rudd P.M. Cristofanilli M. Levels of specific serum N-glycans identify breast cancer patients with higher circulating tumor cell counts.Ann. Oncol. 2011; 22: 1113-1119Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). An alternative method of separation that has been applied in glycan profiling is capillary (gel) electrophoresis, which may be performed on DNA sequencing equipment to allow high-throughput multiplexed analysis (48An H.J. Franz A.H. Lebrilla C.B. Improved capillary electrophoretic separation and mass spectrometric detection of olig