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Isomeric complexity of glycosylation documented by MSn

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Abstract

Re-analysis of two breast cancer cell lines, MCF-7 and MDA-MB-231, has shown multiple isomeric structures exposed by sequential mass spectrometry, MSn. Several released glycan compositions were re-evaluated, which indicated variations in polylactosamine and fucosylation structures. Probable isomer numbers, when considering both stereo and structural entities, are significant and the varying types are mentioned. The structural isomers of linkage position are most frequently considered, while stereo isomers are usually assumed from biosynthetic data. Evaluation of any new sample should be cautious and merits careful attention to empirical data. While isomers are usually considered a chromatographic problem (e.g., LCMS, IMMS) and most frequently considered a separations problem, such results will always be challenged by identification and documentation. MSn data provide a direct spatial solution that includes spectral data for characterization (mass and abundance) supported by a universal library match feature.

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Abbreviations

CID:

Collision-induced disassociation

IM:

Ion mobility

LC:

Liquid chromatography

MSn :

Sequential mass spectrometry

References

  1. Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. 1999;1473(1):4–8.

    Article  CAS  Google Scholar 

  2. Hakomori S. Traveling for the glycosphingolipid path. Glycoconj J. 2000;17(7–9):627–47.

    Article  CAS  Google Scholar 

  3. Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–64. doi:10.1146/annurev.bi.54.070185.003215.

    Article  CAS  Google Scholar 

  4. Spiro RG. Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology. 2002;12(4):43R–56R.

    Article  CAS  Google Scholar 

  5. Stanley P. Golgi glycosylation. Cold Spring Harb Perspect Biol. 2011;3(4). doi: 10.1101/cshperspect.a005199.

  6. Dennis JW, Granovsky M, Warren CE. Protein glycosylation in development and disease. Bioessays. 1999;21(5):412–21. doi:10.1002/(SICI)1521-1878(199905)21:5<412::AID-BIES8>3.0.CO;2-5.

    Article  CAS  Google Scholar 

  7. Dennis JW, Granovsky M, Warren CE. Glycoprotein glycosylation and cancer progression. Biochim Biophys Acta. 1999;1473(1):21–34.

    Article  CAS  Google Scholar 

  8. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochim Biophys Acta. 1999;1473(1):67–95.

    Article  CAS  Google Scholar 

  9. Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci U S A. 2002;99(16):10231–3. doi:10.1073/pnas.172380699.

    Article  CAS  Google Scholar 

  10. Byrd JC, Bresalier RS. Mucins and mucin binding proteins in colorectal cancer. Cancer Metastasis Rev. 2004;23(1–2):77–99.

    Article  CAS  Google Scholar 

  11. Kannagi R, Izawa M, Koike T, Miyazaki K, Kimura N. Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis. Cancer Sci. 2004;95(5):377–84.

    Article  CAS  Google Scholar 

  12. Ono M, Hakomori S. Glycosylation defining cancer cell motility and invasiveness. Glycoconj J. 2004;20(1):71–8. doi:10.1023/B:GLYC.0000018019.22070.7d.

    Article  CAS  Google Scholar 

  13. Jacobs PP, Sackstein R. CD44 and HCELL: preventing hematogenous metastasis at step 1. FEBS Lett. 2011. doi:10.1016/j.febslet.2011.07.039.

    Google Scholar 

  14. Hanley WD, Napier SL, Burdick MM, Schnaar RL, Sackstein R, Konstantopoulos K. Variant isoforms of CD44 are P- and L-selectin ligands on colon carcinoma cells. FASEB J. 2006;20(2):337–9. doi:10.1096/fj.05-4574fje.

    CAS  Google Scholar 

  15. Drake PM, Cho W, Li B, Prakobphol A, Johansen E, Anderson NL, et al. Sweetening the pot: adding glycosylation to the biomarker discovery equation. Clin Chem. 2010;56(2):223–36. doi:10.1373/clinchem.2009.136333.

    Article  CAS  Google Scholar 

  16. Lebrilla CB, An HJ. The prospects of glycan biomarkers for the diagnosis of diseases. Mol Biosyst. 2009;5(1):17–20. doi:10.1039/b811781k.

    Article  CAS  Google Scholar 

  17. Packer NH, von der Lieth CW, Aoki-Kinoshita KF, Lebrilla CB, Paulson JC, Raman R, et al. 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(1):8–20. doi:10.1002/pmic.200700917.

    Article  CAS  Google Scholar 

  18. Goldman R, Ressom HW, Varghese RS, Goldman L, Bascug G, Loffredo CA, et al. Detection of hepatocellular carcinoma using glycomic analysis. Clin Cancer Res. 2009;15(5):1808–13. doi:10.1158/1078-0432.CCR-07-5261.

    Article  CAS  Google Scholar 

  19. Mechref Y, Hussein A, Bekesova S, Pungpapong V, Zhang M, Dobrolecki LE, et al. Quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets. J Proteome Res. 2009;8(6):2656–66. doi:10.1021/pr8008385.

    Article  CAS  Google Scholar 

  20. Leiserowitz GS, Lebrilla C, Miyamoto S, An HJ, Duong H, Kirmiz C, et al. Glycomics analysis of serum: a potential new biomarker for ovarian cancer? Int J Gynecol Cancer. 2008;18(3):470–5. doi:10.1111/j.1525-1438.2007.01028.x.

    Article  CAS  Google Scholar 

  21. Laine RA. A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: the Isomer Barrier to development of single-method saccharide sequencing or synthesis systems. Glycobiology. 1994;4(6):759–67.

    Article  CAS  Google Scholar 

  22. Cummings RD. The repertoire of glycan determinants in the human glycome. Mol Biosyst. 2009;5(10):1087–104. doi:10.1039/b907931a.

    Article  CAS  Google Scholar 

  23. McLafferty FW. Interpretation of Mass Spectra. 2nd ed. Reading, MA: W. A. Benjamin, Inc.; 1973.

    Google Scholar 

  24. Zaia J. Mass spectrometry and glycomics. OMICS. 2010;14(4):401–18. doi:10.1089/omi.2009.0146.

    Article  CAS  Google Scholar 

  25. Tao S, Huang Y, Boyes BE, Orlando R. Liquid chromatography-selected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers. Anal Chem. 2014;86(21):10584–90. doi:10.1021/ac5020996.

    Article  CAS  Google Scholar 

  26. Kolarich D, Windwarder M, Alagesan K, Altmann F. Isomer-specific analysis of released N-glycans by LC-ESI MS/MS with porous graphitized carbon. Methods Mol Biol. 2015;1321:427–35. doi:10.1007/978-1-4939-2760-9_29.

    Article  Google Scholar 

  27. Stavenhagen K, Kolarich D, Wuhrer M. Clinical glycomics employing graphitized carbon liquid chromatography-mass spectrometry. Chromatographia. 2015;78(5–6):307–20. doi:10.1007/s10337-014-2813-7.

    Article  CAS  Google Scholar 

  28. Michael C, Rizzi AM. Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase. J Chromatogr A. 2015;1383:88–95. doi:10.1016/j.chroma.2015.01.028.

    Article  CAS  Google Scholar 

  29. Gaye MM, Kurulugama R, Clemmer DE. Investigating carbohydrate isomers by IMS-CID-IMS-MS: precursor and fragment ion cross-sections. Analyst. 2015;140(20):6922–32. doi:10.1039/c5an00840a.

    Article  CAS  Google Scholar 

  30. Hoffmann W, Hofmann J, Pagel K. Energy-resolved ion mobility-mass spectrometry—a concept to improve the separation of isomeric carbohydrates. J Am Soc Mass Spectrom. 2014;25(3):471–9. doi:10.1007/s13361-013-0780-0.

    Article  CAS  Google Scholar 

  31. Huang Y, Dodds ED. Discrimination of isomeric carbohydrates as the electron transfer products of group II cation adducts by ion mobility spectrometry and tandem mass spectrometry. Anal Chem. 2015;87(11):5664–8. doi:10.1021/acs.analchem.5b00759.

    Article  CAS  Google Scholar 

  32. Huang Y, Dodds ED. Ion-neutral collisional cross sections of carbohydrate isomers as divalent cation adducts and their electron transfer products. Analyst. 2015;140(20):6912–21. doi:10.1039/c5an01093d.

    Article  CAS  Google Scholar 

  33. Lee S, Valentine SJ, Reilly JP, Clemmer DE. Analyzing a Mixture of Disaccharides by IMS-VUVPD-MS. Int J Mass Spectrom. 2012;309:161–7. doi:10.1016/j.ijms.2011.09.013.

    Article  CAS  Google Scholar 

  34. Li H, Bendiak B, Kaplan K, Davis E, Siems WF, Hill Jr HH. Evaluation of ion mobility-mass spectrometry for determining the isomeric heterogeneity of oligosaccharide-alditols derived from bovine submaxillary mucin. Int J Mass Spectrom. 2013;352:9–18. doi:10.1016/j.ijms.2013.07.015.

    Article  CAS  Google Scholar 

  35. Li H, Bendiak B, Siems WF, Gang DR, Hill Jr HH. Ion mobility mass spectrometry analysis of isomeric disaccharide precursor, product and cluster ions. Rapid Commun Mass Spectrom. 2013;27(23):2699–709. doi:10.1002/rcm.6720.

    Article  CAS  Google Scholar 

  36. Pu Y, Ridgeway ME, Glaskin RS, Park MA, Costello CE, Lin C. Separation and identification of isomeric glycans by selected accumulation-trapped ion mobility spectrometry-electron activated dissociation tandem mass spectrometry. Anal Chem. 2016;88(7):3440–3. doi:10.1021/acs.analchem.6b00041.

    Article  CAS  Google Scholar 

  37. Guerrero A, Lebrilla CB. New strategies for resolving oligosaccharide isomers by exploiting mechanistic and thermochemical aspects of fragment ion formation. Int J Mass Spectrom. 2013;354–355:19–25. doi:10.1016/j.ijms.2013.05.002.

    Article  Google Scholar 

  38. Han L, Costello CE. Electron transfer dissociation of milk oligosaccharides. J Am Soc Mass Spectrom. 2011;22(6):997–1013. doi:10.1007/s13361-011-0117-9.

    Article  CAS  Google Scholar 

  39. Jovanovic M, Tyldesley-Worster R, Pohlentz G, Peter-Katalinic J. MALDI Q-TOF CID MS for diagnostic ion screening of human milk oligosaccharide samples. Int J Mol Sci. 2014;15(4):6527–43. doi:10.3390/ijms15046527.

    Article  CAS  Google Scholar 

  40. Konda C, Londry FA, Bendiak B, Xia Y. Assignment of the stereochemistry and anomeric configuration of sugars within oligosaccharides via overlapping disaccharide ladders using MS(n). J Am Soc Mass Spectrom. 2014;25(8):1441–50. doi:10.1007/s13361-014-0881-4.

    Article  CAS  Google Scholar 

  41. Nagy G, Pohl NL. Monosaccharide identification as a first step toward de novo carbohydrate sequencing: mass spectrometry strategy for the identification and differentiation of diastereomeric and enantiomeric pentose isomers. Anal Chem. 2015;87(8):4566–71. doi:10.1021/acs.analchem.5b00760.

    Article  CAS  Google Scholar 

  42. Nagy G, Pohl NL. Complete hexose isomer identification with mass spectrometry. J Am Soc Mass Spectrom. 2015;26(4):677–85. doi:10.1007/s13361-014-1072-z.

    Article  CAS  Google Scholar 

  43. Tan Y, Polfer NC. Linkage and anomeric differentiation in trisaccharides by sequential fragmentation and variable-wavelength infrared photodissociation. J Am Soc Mass Spectrom. 2015;26(2):359–68. doi:10.1007/s13361-014-1025-6.

    Article  CAS  Google Scholar 

  44. Garner B, Priestman DA, Stocker R, Harvey DJ, Butters TD, Platt FM. Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice. J Lipid Res. 2002;43(2):205–14.

    CAS  Google Scholar 

  45. Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo. J Biol Chem. 2007;282(12):9127–42. doi:10.1074/jbc.M606711200.

    Article  CAS  Google Scholar 

  46. Canis K, McKinnon TA, Nowak A, Panico M, Morris HR, Laffan M, et al. The plasma von Willebrand factor O-glycome comprises a surprising variety of structures including ABH antigens and disialosyl motifs. J Thromb Haemost. 2010;8(1):137–45. doi:10.1111/j.1538-7836.2009.03665.x.

    Article  CAS  Google Scholar 

  47. Alley Jr WR, Madera M, Mechref Y, Novotny MV. Chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated N-linked glycans: a potential methodology for cancer-biomarker discovery. Anal Chem. 2010;82(12):5095–106. doi:10.1021/ac100131e.

    Article  CAS  Google Scholar 

  48. Carlson DM. Oligosaccharides isolated from pig submaxillary mucin. J Biol Chem. 1966;241(12):2984–6.

    CAS  Google Scholar 

  49. Kang P, Mechref Y, Klouckova I, Novotny MV. Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun Mass Spectrom. 2005;19(23):3421–8. doi:10.1002/rcm.2210.

    Article  CAS  Google Scholar 

  50. Ashline DJ, Hanneman AJ, Zhang H, Reinhold VN. Structural documentation of glycan epitopes: sequential mass spectrometry and spectral matching. J Am Soc Mass Spectrom. 2014;25(3):444–53. doi:10.1007/s13361-013-0776-9.

    Article  CAS  Google Scholar 

  51. Ashline DJ, Yu Y, Lasanajak Y, Song X, Hu L, Ramani S, et al. Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses. Mol Cell Proteomics. 2014;13(11):2961–74. doi:10.1074/mcp.M114.039925.

    Article  CAS  Google Scholar 

  52. Nomenclature of glycolipids. Carbohydr Res. 1998;312:167–75.

  53. Ashline D, Singh S, Hanneman A, Reinhold V. Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn. Anal Chem. 2005;77(19):6250–62. doi:10.1021/ac050724z.

    Article  CAS  Google Scholar 

  54. Li Y, Teneberg S, Thapa P, Bendelac A, Levery SB, Zhou D. Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry. Glycobiology. 2008;18(2):158–65. doi:10.1093/glycob/cwm129.

    Article  CAS  Google Scholar 

  55. Powlesland AS, Hitchen PG, Parry S, Graham SA, Barrio MM, Elola MT, et al. Targeted glycoproteomic identification of cancer cell glycosylation. Glycobiology. 2009;19(8):899–909. doi:10.1093/glycob/cwp065.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported, in part, by a Program of Excellence in Glycosciences grant (P01 HL107146, PI Robert Sackstein) and Glycan Connections, LLC, Lee NH.

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Authors and Affiliations

  1. The Glycomics Center, Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, 35 Colovos Road, Durham, NH, 03824, USA

    David J. Ashline, Hailong Zhang & Vernon N. Reinhold

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  1. David J. Ashline
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  2. Hailong Zhang
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  3. Vernon N. Reinhold
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Correspondence to Vernon N. Reinhold.

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Published in the topical collection Glycomics, Glycoproteomics and Allied Topics with guest editors Yehia Mechref and David Muddiman.

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Ashline, D.J., Zhang, H. & Reinhold, V.N. Isomeric complexity of glycosylation documented by MSn . Anal Bioanal Chem 409, 439–451 (2017). https://doi.org/10.1007/s00216-016-0018-7

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