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Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode

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Abstract

Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)— with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica— with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.

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References

  1. Varki A. Biological roles of glycans. Glycobiology. 2017;27(1):3–49. https://doi.org/10.1093/glycob/cww086.

    Article  CAS  PubMed  Google Scholar 

  2. Dong X, Mondello S, Kobeissy F, Ferri R, Mechref Y. Serum glycomics profiling of patients with primary restless legs syndrome using LC-MS/MS. J Proteome Res. 2020;19(8):2933–41. https://doi.org/10.1021/acs.jproteome.9b00549.

    Article  CAS  PubMed  Google Scholar 

  3. Peng WJ, Goli M, Mirzaei P, Mechref Y. Revealing the biological attributes of N-glycan isomers in breast cancer brain metastasis using porous graphitic carbon (PGC) liquid chromatography-tandem mass spectrometry (LC-MS/MS). J Proteome Res. 2019;18(10):3731–40. https://doi.org/10.1021/acs.jproteome.9b00429.

    Article  CAS  PubMed  Google Scholar 

  4. Duivelshof BL, Jiskoot W, Beck A, Veuthey JL, Guillarme D, D'Atri V. Glycosylation of biosimilars: recent advances in analytical characterization and clinical implications. Anal Chim Acta. 2019;1089:1–18. https://doi.org/10.1016/j.aca.2019.08.044.

    Article  CAS  PubMed  Google Scholar 

  5. Shajahan A, Heiss C, Ishihara M, Azadi P. Glycomic and glycoproteomic analysis of glycoproteins-a tutorial. Anal Bioanal Chem. 2017;409(19):4483–505. https://doi.org/10.1007/s00216-017-0406-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kozlik P, Sanda M, Goldman R. Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides. J Chromatogr A. 2017;1519:152–5. https://doi.org/10.1016/j.chroma.2017.08.066.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kozlik P, Goldman R, Sanda M. Study of structure-dependent chromatographic behavior of glycopeptides using reversed phase nanoLC. Electrophoresis. 2017;38(17):2193–9. https://doi.org/10.1002/elps.201600547.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Badgett MJ, Boyes B, Orlando R. Predicting the retention behavior of specific O-linked glycopeptides. J Biomol Tech. 2017;28(3):122–6. https://doi.org/10.7171/jbt.17-2803-003.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Badgett MJ, Boyes B, Orlando R. Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry. J Chromatogr A. 2018;1537:58–65. https://doi.org/10.1016/j.chroma.2017.12.055.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. D'Atri V, Fekete S, Stoll D, Lauber M, Beck A, Guillarme D. Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry. J Chromatogr B. 2018;1080:37–41. https://doi.org/10.1016/j.jchromb.2018.02.026.

    Article  CAS  Google Scholar 

  11. D'Atri V, Novakova L, Fekete S, Stoll D, Lauber M, Beck A, et al. Orthogonal middle-up approaches for characterization of the glycan heterogeneity of etanercept by hydrophilic interaction chromatography coupled to high-resolution mass spectrometry. Anal Chem. 2019;91(1):873–80. https://doi.org/10.1021/acs.analchem.8b03584.

    Article  CAS  PubMed  Google Scholar 

  12. Huang Y, Nie Y, Boyes B, Orlando R. Resolving isomeric glycopeptide glycoforms with hydrophilic interaction chromatography (HILIC). J Biomol Tech. 2016;27(3):98–104. https://doi.org/10.7171/jbt.16-2703-003.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Kozlik P, Goldman R, Sanda M. Hydrophilic interaction liquid chromatography in the separation of glycopeptides and their isomers. Anal Bioanal Chem. 2018;410(20):5001–8. https://doi.org/10.1007/s00216-018-1150-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Molnarova K, Kozlik P. Comparison of different HILIC stationary phases in the separation of hemopexin and immunoglobulin G glycopeptides and their isomers. Molecules. 2020;25(20):13. https://doi.org/10.3390/molecules25204655.

    Article  CAS  Google Scholar 

  15. Kartsova LA, Bessonova EA, Somova VD. Hydrophilic interaction chromatography. J Anal Chem. 2019;74(5):415–24. https://doi.org/10.1134/s1061934819050058.

    Article  CAS  Google Scholar 

  16. Jandera P, Janas P. Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review. Anal Chim Acta. 2017;967:12–32. https://doi.org/10.1016/j.aca.2017.01.060.

    Article  CAS  PubMed  Google Scholar 

  17. McCalley DV. Understanding and manipulating the separation in hydrophilic interaction liquid chromatography. J Chromatogr A. 2017;1523:49–71. https://doi.org/10.1016/j.chroma.2017.06.026.

    Article  CAS  PubMed  Google Scholar 

  18. Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Anal Bioanal Chem. 2012;402(1):231–47. https://doi.org/10.1007/s00216-011-5308-5.

    Article  CAS  PubMed  Google Scholar 

  19. Pedrali A, Tengattini S, Marrubini G, Bavaro T, Hemstrom P, Massolini G, et al. Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography. Molecules. 2014;19(7):9070–88. https://doi.org/10.3390/molecules19079070.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Takegawa Y, Deguchi K, Ito H, Keira T, Nakagawa H, Nishimura SI. Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography. J Sep Sci. 2006;29(16):2533–40. https://doi.org/10.1002/jssc.200600133.

    Article  CAS  PubMed  Google Scholar 

  21. Tengattini S, Dominguez-Vega E, Temporini C, Bavaro T, Rinaldi F, Piubelli L, et al. Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins. Anal Chim Acta. 2017;981:94–105. https://doi.org/10.1016/j.aca.2017.05.020.

    Article  CAS  PubMed  Google Scholar 

  22. Lauber MA, Yu YQ, Brousmiche DW, Hua ZM, Koza SM, Magnelli P, et al. Rapid preparation of released N-glycans for HILIC analysis using a labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Anal Chem. 2015;87(10):5401–9. https://doi.org/10.1021/acs.analchem.5b00758.

    Article  CAS  PubMed  Google Scholar 

  23. Romera-Torres A, Romero-Gonzalez R, Vidal JLM, Frenich AG. Comprehensive tropane alkaloids analysis and retrospective screening of contaminants in honey samples using liquid chromatography-high resolution mass spectrometry (Orbitrap). Food Res Int. 2020;133:9. https://doi.org/10.1016/j.foodres.2020.109130.

    Article  CAS  Google Scholar 

  24. Pavlaki A, Begou O, Deda O, Farmaki E, Dotis J, Gika H, et al. Serum-targeted HILIC-MS metabolomics-based analysis in infants with ureteropelvic junction obstruction. J Proteome Res. 2020;19(6):2294–303. https://doi.org/10.1021/acs.jproteome.9b00855.

    Article  CAS  PubMed  Google Scholar 

  25. Manzano-Sánchez L, Martínez-Martínez JA, Domínguez I, Vidal JLM, Frenich AG, Romero-González R. Development and application of a novel pluri-residue method to determine polar pesticides in fruits and vegetables through liquid chromatography high resolution mass spectrometry. Foods. 2020;9(5). https://doi.org/10.3390/foods9050553.

  26. de Haan N, Falck D, Wuhrer M. Monitoring of immunoglobulin N- and O-glycosylation in health and disease. Glycobiology. 2020;30(4):226–40. https://doi.org/10.1093/glycob/cwz048.

    Article  CAS  PubMed  Google Scholar 

  27. Momcilovic A, de Haan N, Ederveen ALH, Bondt A, Koeleman CAM, Falck D, et al. Simultaneous immunoglobulin A and G glycopeptide profiling for high-throughput applications. Anal Chem. 2020;92(6):4518–26. https://doi.org/10.1021/acs.analchem.9b05722.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vreeker GCM, Wuhrer M. Reversed-phase separation methods for glycan analysis. Anal Bioanal Chem. 2017;409(2):359–78. https://doi.org/10.1007/s00216-016-0073-0.

    Article  CAS  PubMed  Google Scholar 

  29. Kadlecova Z, Kalikova K, Folprechtova D, Tesarova E, Gilar M. Method for evaluation of ionic interactions in liquid chromatography. J Chromatogr A. 1625;2020:7. https://doi.org/10.1016/j.chroma.2020.461301.

    Article  CAS  Google Scholar 

  30. Badgett MJ, Mize E, Fletcher T, Boyes B, Orlando R. Predicting the HILIC retention behavior of the N-linked glycopeptides produced by trypsin digestion of immunoglobulin Gs (IgGs). J Biomol Tech. 2018;29(4):98–104. https://doi.org/10.7171/jbt.18-2904-002.

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

The present work was supported by the Czech Science Foundation, Grant No 19-18005Y. The research was supported in part by the Charles University project SVV260560 and Ministry of Education, Youth and Sports of the Czech Republic (LTC20078 in frame of the COST Action CA18103 INNOGLY).

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43, Prague 2, Czech Republic

    Katarina Molnarova, Ales Duris & Petr Kozlik

  2. Department of Biochemistry, Faculty of Science, Charles University, 128 00, Prague 2, Czech Republic

    Tomas Jecmen

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  1. Katarina Molnarova
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Correspondence to Petr Kozlik.

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Molnarova, K., Duris, A., Jecmen, T. et al. Comparison of human IgG glycopeptides separation using mixed-mode hydrophilic interaction/ion-exchange liquid chromatography and reversed-phase mode. Anal Bioanal Chem 413, 4321–4328 (2021). https://doi.org/10.1007/s00216-021-03388-3

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