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Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow

Abstract

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.

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Fig. 1: Schematic of the PCMF setup.
Fig. 2: Comparison of the spray stability during elution when using nano-PGC-LC-ESI-MS(/MS) in negative ion mode.
Fig. 3: Comparison of the BPC of eluted N- and O-glycans released from bovine fetuin before (a) and after (b) PCMF supplementation.
Fig. 4: Comparison of the EIC of selected masses corresponding to N- and O-glycans released from bovine fetuin before (a) and after (b) PCMF supplementation.

Abbreviations

ABC:

ammonium bicarbonate

BPC:

base peak chromatogram

C18:

octadecyl carbon chain

DTT:

dithiothreitol

EIC:

extracted ion chromatogram

ESI:

electrospray ionization

HCD:

higher-energy collisional dissociation

HILIC:

hydrophilic interaction liquid chromatography

HPLC:

high-performance liquid chromatography

IAA:

iodoacetamide

ID:

inner diameter

KOH:

potassium hydroxide

LC-MS:

liquid chromatography coupled to mass spectrometry

MS:

mass spectrometry

MS/MS:

tandem mass spectrometry

NaBH4 :

sodium borohydride

OD:

outer diameter

PCMF:

post-column make-up flow

PGC:

porous graphitized carbon

UPLC:

ultra performance liquid chromatography

References

  1. 1.

    Varki, A., Hart, G.W.: Essentials of Glycobiology. Cold Spring Harbor Laboratory Press (2017)

  2. 2.

    Varki, A.: Biological roles of glycans. Glycobiology. 27(1), 3–49 (2017)

  3. 3.

    Helenius, A., Aebi, M.: Intracellular functions of N-linked glycans. Science. 291(5512), 2364–2369 (2001)

  4. 4.

    Varki, A.: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 3(2), 97–130 (1993)

  5. 5.

    Moremen, K.W., Tiemeyer, M., Nairn, A.V.: Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol. 13(7), 448–462 (2012). https://doi.org/10.1038/nrm3383

  6. 6.

    Woods, R.J.: Three-dimensional structures of oligosaccharides. Curr. Opin. Struct. Biol. 5(5), 591–598 (1995)

  7. 7.

    Wang, J.-R., Gao, W.-N., Grimm, R., Jiang, S., Liang, Y., Ye, H., Li, Z.-G., Yau, L.-F., Huang, H., Liu, J.: A method to identify trace sulfated IgG N-glycans as biomarkers for rheumatoid arthritis. Nat. Commun. 8(1), 631 (2017)

  8. 8.

    Pomin, V.H.: Sulfated glycans in inflammation. Eur. J. Med. Chem. 92, 353–369 (2015)

  9. 9.

    Yoshimura, T., Hayashi, A., Handa-Narumi, M., Yagi, H., Ohno, N., Koike, T., Yamaguchi, Y., Uchimura, K., Kadomatsu, K., Sedzik, J.: GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system. Sci. Rep. 7, 42257 (2017)

  10. 10.

    Takashiba, M., Chiba, Y., Jigami, Y.: Identification of phosphorylation sites in N-linked glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 78(14), 5208–5213 (2006)

  11. 11.

    Zhang, L., Luo, S., Zhang, B.: Glycan analysis of therapeutic glycoproteins. MAbs. 8(2), 205–215 (2016). https://doi.org/10.1080/19420862.2015.1117719

  12. 12.

    Aich, U., Lakbub, J., Liu, A.: State-of-the-art technologies for rapid and high-throughput sample preparation and analysis of N-glycans from antibodies. Electrophoresis. 37(11), 1468–1488 (2016). https://doi.org/10.1002/elps.201500551

  13. 13.

    Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., Dwek, R.A.: A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240(2), 210–226 (1996). https://doi.org/10.1006/abio.1996.0351

  14. 14.

    Rudd, P.M., Dwek, R.A.: Rapid, sensitive sequencing of oligosaccharides from glycoproteins. Curr. Opin. Biotechnol. 8(4), 488–497 (1997)

  15. 15.

    Ahn, J., Bones, J., Yu, Y.Q., Rudd, P.M., Gilar, M.: Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent. Journal Chromatogr. B, Anal Technol Biomed Life Sci. 878(3–4), 403–408 (2010). https://doi.org/10.1016/j.jchromb.2009.12.013

  16. 16.

    Callewaert, N., Geysens, S., Molemans, P., Contreras, R.: Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology. 11(4), 275–281 (2001). https://doi.org/10.1093/glycob/11.4.275

  17. 17.

    Schwarzer, J., Rapp, E., Reichl, U.: N-glycan analysis by CGE-LIF: profiling influenza a virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis. 29(20), 4203–4214 (2008). https://doi.org/10.1002/elps.200800042

  18. 18.

    Schwarzer, J., Rapp, E., Hennig, R., Genzel, Y., Jordan, I., Sandig, V., Reichl, U.: Glycan analysis in cell culture-based influenza vaccine production: influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin. Vaccine. 27(32), 4325–4336 (2009). https://doi.org/10.1016/j.vaccine.2009.04.076

  19. 19.

    Ruhaak, L.R., Hennig, R., Huhn, C., Borowiak, M., Dolhain, R.J., Deelder, A.M., Rapp, E., Wuhrer, M.: Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J. Proteome Res. 9(12), 6655–6664 (2010)

  20. 20.

    Hennig, R., Rapp, E., Kottler, R., Cajic, S., Borowiak, M., Reichl, U.: N-Glycosylation fingerprinting of viral glycoproteins by xCGE-LIF. Methods Mol. Biol. 1331, 123–143 (2015). https://doi.org/10.1007/978-1-4939-2874-3_8

  21. 21.

    Wuhrer, M., de Boer, A.R., Deelder, A.M.: Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry. Mass Spectrom. Rev. 28(2), 192–206 (2009). https://doi.org/10.1002/mas.20195

  22. 22.

    Jensen, P.H., Karlsson, N.G., Kolarich, D., Packer, N.H.: Structural analysis of N- and O-glycans released from glycoproteins. Nat. Protoc. 7(7), 1299–1310 (2012). https://doi.org/10.1038/nprot.2012.063

  23. 23.

    Everest-Dass, A.V., Kolarich, D., Campbell, M.P., Packer, N.H.: Tandem mass spectra of glycan substructures enable the multistage mass spectrometric identification of determinants on oligosaccharides. Rapid Commun Mass Spectrom : RCM. 27(9), 931–939 (2013). https://doi.org/10.1002/rcm.6527

  24. 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. 86(12), 5784–5793 (2014). https://doi.org/10.1021/ac500335t

  25. 25.

    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. 1321, 427–435 (2015). https://doi.org/10.1007/978-1-4939-2760-9_29

  26. 26.

    Wuhrer, M., Deelder, A.M., Hokke, C.H.: Protein glycosylation analysis by liquid chromatography-mass spectrometry. Journal Chromatogr. B, Anal Technol Biomed Life Sci. 825(2), 124–133 (2005). https://doi.org/10.1016/j.jchromb.2005.01.030

  27. 27.

    Reiding, K.R., Lonardi, E., Hipgrave Ederveen, A.L., Wuhrer, M.: Ethyl esterification for MALDI-MS analysis of protein glycosylation. Methods Mol. Biol. 1394, 151–162 (2016). https://doi.org/10.1007/978-1-4939-3341-9_11

  28. 28.

    Shubhakar, A., Kozak, R.P., Reiding, K.R., Royle, L., Spencer, D.I., Fernandes, D.L., Wuhrer, M.: Automated high-throughput Permethylation for glycosylation analysis of biologics using MALDI-TOF-MS. Anal. Chem. 88(17), 8562–8569 (2016). https://doi.org/10.1021/acs.analchem.6b01639

  29. 29.

    Kottler, R., Mank, M., Hennig, R., Muller-Werner, B., Stahl, B., Reichl, U., Rapp, E.: Development of a high-throughput glycoanalysis method for the characterization of oligosaccharides in human milk utilizing multiplexed capillary gel electrophoresis with laser-induced fluorescence detection. Electrophoresis. 34(16), 2323–2336 (2013). https://doi.org/10.1002/elps.201300016

  30. 30.

    Rodig, J.V., Rapp, E., Bohne, J., Kampe, M., Kaffka, H., Bock, A., Genzel, Y., Reichl, U.: Impact of cultivation conditions on N-glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture. Biotechnol. Bioeng. 110(6), 1691–1703 (2013). https://doi.org/10.1002/bit.24834

  31. 31.

    Hutter, J., Rodig, J.V., Hoper, D., Seeberger, P.H., Reichl, U., Rapp, E., Lepenies, B.: Toward animal cell culture-based influenza vaccine design: viral hemagglutinin N-glycosylation markedly impacts immunogenicity. J. Immunol. 190(1), 220–230 (2013). https://doi.org/10.4049/jimmunol.1201060

  32. 32.

    Hennig, R., Cajic, S., Borowiak, M., Hoffmann, M., Kottler, R., Reichl, U., Rapp, E.: Towards personalized diagnostics via longitudinal study of the human plasma N-glycome. Biochim. Biophys. Acta. 1860(8), 1728–1738 (2016). https://doi.org/10.1016/j.bbagen.2016.03.035

  33. 33.

    Thiesler, C.T., Cajic, S., Hoffmann, D., Thiel, C., van Diepen, L., Hennig, R., Sgodda, M., Weibetamann, R., Reichl, U., Steinemann, D., Diekmann, U., Huber, N.M., Oberbeck, A., Cantz, T., Kuss, A.W., Korner, C., Schambach, A., Rapp, E., Buettner, F.F.: Glycomic characterization of induced pluripotent stem cells derived from a patient suffering from Phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG). Mol Cell Proteomics : MCP. 15(4), 1435–1452 (2016). https://doi.org/10.1074/mcp.M115.054122

  34. 34.

    Konze, S.A., Cajic, S., Oberbeck, A., Hennig, R., Pich, A., Rapp, E., Buettner, F.F.R.: Quantitative assessment of sialo-glycoproteins and N-Glycans during Cardiomyogenic differentiation of human induced pluripotent stem cells. Chembiochem. 18(13), 1317–1331 (2017). https://doi.org/10.1002/cbic.201700100

  35. 35.

    Ciucanu, I.: Per-O-methylation reaction for structural analysis of carbohydrates by mass spectrometry. Anal. Chim. Acta. 576(2), 147–155 (2006). https://doi.org/10.1016/j.aca.2006.06.009

  36. 36.

    Morelle, W., Michalski, J.C.: Analysis of protein glycosylation by mass spectrometry. Nat. Protoc. 2(7), 1585–1602 (2007). https://doi.org/10.1038/nprot.2007.227

  37. 37.

    Harvey, D.J.: Electrospray mass spectrometry and collision-induced fragmentation of 2-aminobenzamide-labelled neutral N-linked glycans. Analyst. 125(4), 609–617 (2000). https://doi.org/10.1039/a908997g

  38. 38.

    Melmer, M., Stangler, T., Schiefermeier, M., Brunner, W., Toll, H., Rupprechter, A., Lindner, W., Premstaller, A.: HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals. Anal. Bioanal. Chem. 398(2), 905–914 (2010). https://doi.org/10.1007/s00216-010-3988-x

  39. 39.

    Stockmann, H., O'Flaherty, R., Adamczyk, B., Saldova, R., Rudd, P.M.: Automated, high-throughput serum glycoprofiling platform. Integr Biol (Camb). 7(9), 1026–1032 (2015). https://doi.org/10.1039/c5ib00130g

  40. 40.

    Anumula, K.R., Dhume, S.T.: High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiology. 8(7), 685–694 (1998)

  41. 41.

    Klapoetke, S., Zhang, J., Becht, S., Gu, X., Ding, X.: The evaluation of a novel approach for the profiling and identification of N-linked glycan with a procainamide tag by HPLC with fluorescent and mass spectrometric detection. J. Pharm. Biomed. Anal. 53(3), 315–324 (2010). https://doi.org/10.1016/j.jpba.2010.03.045

  42. 42.

    Lauber, M.A., Yu, Y.Q., Brousmiche, D.W., Hua, Z., Koza, S.M., Magnelli, P., Guthrie, E., Taron, C.H., Fountain, K.J.: Rapid preparation of released N-Glycans for HILIC analysis using a labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Anal. Chem. 87(10), 5401–5409 (2015). https://doi.org/10.1021/acs.analchem.5b00758

  43. 43.

    Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., Takahashi, N.: Analyses of N-linked oligosaccharides using a two-dimensional mapping technique. Anal. Biochem. 171(1), 73–90 (1988)

  44. 44.

    Marino, K., Bones, J., Kattla, J.J., Rudd, P.M.: A systematic approach to protein glycosylation analysis: a path through the maze. Nat. Chem. Biol. 6(10), 713–723 (2010). https://doi.org/10.1038/Nchembio.437

  45. 45.

    Nakano, M., Saldanha, R., Gobel, A., Kavallaris, M., Packer, N.H.: Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells. Mol. Cell. Proteomics : MCP. 10(11), M111 009001 (2011). https://doi.org/10.1074/mcp.M111.009001

  46. 46.

    Everest-Dass, A.V., Jin, D.Y., Thaysen-Andersen, M., Nevalainen, H., Kolarich, D., Packer, N.H.: Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: innate protection against infection by Candida albicans. Glycobiology. 22(11), 1465–1479 (2012). https://doi.org/10.1093/glycob/cws112

  47. 47.

    Nguyen-Khuong, T., Everest-Dass, A.V., Kautto, L., Zhao, Z.J., Willcox, M.D.P., Packer, N.H.: Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy. Glycobiology. 25(3), 269–283 (2015). https://doi.org/10.1093/glycob/cwu108

  48. 48.

    Ashwood, C., Lin, C.H., Thaysen-Andersen, M., Packer, N.H.: Discrimination of isomers of released N- and O-Glycans using diagnostic product ions in negative ion PGC-LC-ESI-MS/MS. J. Am. Soc. Mass Spectrom. 29(6), 1194–1209 (2018). https://doi.org/10.1007/s13361-018-1932-z

  49. 49.

    Estrella, R.P., Whitelock, J.M., Packer, N.H., Karlsson, N.G.: Graphitized carbon LC− MS characterization of the chondroitin sulfate oligosaccharides of Aggrecan. Anal. Chem. 79(10), 3597–3606 (2007)

  50. 50.

    Schulz, B.L., Sloane, A.J., Robinson, L.J., Prasad, S.S., Lindner, R.A., Robinson, M., Bye, P.T., Nielson, D.W., Harry, J.L., Packer, N.H.: Glycosylation of sputum mucins is altered in cystic fibrosis patients. Glycobiology. 17(7), 698–712 (2007)

  51. 51.

    Thomsson, K.A., Schulz, B.L., Packer, N.H., Karlsson, N.G.: MUC5B glycosylation in human saliva reflects blood group and secretor status. Glycobiology. 15(8), 791–804 (2005)

  52. 52.

    Karlsson, N.G., Schulz, B.L., Packer, N.H.: Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 15(5), 659–672 (2004). https://doi.org/10.1016/j.jasms.2004.01.002

  53. 53.

    Schulz, B.L., Oxley, D., Packer, N.H., Karlsson, N.G.: Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears. Biochem. J. 366(2), 511–520 (2002)

  54. 54.

    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: part 2. Fragmentation of high-mannose N-linked glycans. J. Am. Soc. Mass Spectrom. 16(5), 631–646 (2005). https://doi.org/10.1016/j.jasms.2005.01.005

  55. 55.

    Harvey, D.J.: Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans. J. Am. Soc. Mass Spectrom. 16(5), 647–659 (2005). https://doi.org/10.1016/j.jasms.2005.01.006

  56. 56.

    Harvey, D.J., Royle, L., Radcliffe, C.M., Rudd, P.M., Dwek, R.A.: Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry. Anal. Biochem. 376(1), 44–60 (2008). https://doi.org/10.1016/j.ab.2008.01.025

  57. 57.

    Everest-Dass, A.V., Abrahams, J.L., Kolarich, D., Packer, N.H., Campbell, M.P.: Structural feature ions for distinguishing N- and O-linked glycan isomers by LC-ESI-IT MS/MS. J. Am. Soc. Mass Spectrom. 24(6), 895–906 (2013). https://doi.org/10.1007/s13361-013-0610-4

  58. 58.

    Wilm, M., Mann, M.: Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68(1), 1–8 (1996). https://doi.org/10.1021/ac9509519

  59. 59.

    El-Faramawy, A., Siu, K.M., Thomson, B.A.: Efficiency of nano-electrospray ionization. J. Am. Soc. Mass Spectrom. 16(10), 1702–1707 (2005)

  60. 60.

    Jensen, P.H., Kolarich, D., Packer, N.H.: Mucin-type O-glycosylation - putting the pieces together. FEBS J. 277(1), 81–94 (2010). https://doi.org/10.1111/j.1742-4658.2009.07429.x

  61. 61.

    Wisniewski, J.R., Zougman, A., Nagaraj, N., Mann, M.: Universal sample preparation method for proteome analysis. Nat. Methods. 6(5), 359–362 (2009). https://doi.org/10.1038/nmeth.1322

  62. 62.

    Varki, A., Cummings, R.D., Aebi, M., Packer, N.H., Seeberger, P.H., Esko, J.D., Stanley, P., Hart, G., Darvill, A., Kinoshita, T., Prestegard, J.J., Schnaar, R.L., Freeze, H.H., Marth, J.D., Bertozzi, C.R., Etzler, M.E., Frank, M., Vliegenthart, J.F., Lutteke, T., Perez, S., Bolton, E., Rudd, P., Paulson, J., Kanehisa, M., Toukach, P., Aoki-Kinoshita, K.F., Dell, A., Narimatsu, H., York, W., Taniguchi, N., Kornfeld, S.: Symbol nomenclature for graphical representations of Glycans. Glycobiology. 25(12), 1323–1324 (2015). https://doi.org/10.1093/glycob/cwv091

  63. 63.

    Kawasaki, N., Ohta, M., Hyuga, S., Hyuga, M., Hayakawa, T.: Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin. Anal. Biochem. 285(1), 82–91 (2000). https://doi.org/10.1006/abio.2000.4739

  64. 64.

    Kawasaki, N., Itoh, S., Ohta, M., Hayakawa, T.: Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. Anal. Biochem. 316(1), 15–22 (2003)

  65. 65.

    Karlsson, N.G., Wilson, N.L., Wirth, H.J., Dawes, P., Joshi, H., Packer, N.H.: Negative ion graphitised carbon nano-liquid chromatography/mass spectrometry increases sensitivity for glycoprotein oligosaccharide analysis. Rapid Commun Mass Spectrom : RCM. 18(19), 2282–2292 (2004). https://doi.org/10.1002/rcm.1626

  66. 66.

    Pabst, M., Bondili, J.S., Stadlmann, J., Mach, L., Altmann, F.: Mass + retention time = structure: a strategy for the analysis of N-glycans by carbon LC-ESI-MS and its application to fibrin N-glycans. Anal. Chem. 79(13), 5051–5057 (2007). https://doi.org/10.1021/ac070363i

  67. 67.

    Hershberger, L.W., Callis, J.B., Christian, G.D.: Sub-microliter flow-through Cuvette for fluorescence monitoring of high-performance liquid-chromatographic effluents. Anal. Chem. 51(9), 1444–1446 (1979). https://doi.org/10.1021/ac50045a021

  68. 68.

    Bruins, A.P., Covey, T.R., Henion, J.D.: Ion spray Interface for combined liquid chromatography/atmospheric pressure ionization mass-spectrometry. Anal. Chem. 59(22), 2642–2646 (1987). https://doi.org/10.1021/ac00149a003

  69. 69.

    Straub, R.F., Voyksner, R.D.: Negative-ion formation in electrospray mass-spectrometry. J Am Soc Mass Spectrom. 4(7), 578–587 (1993). https://doi.org/10.1016/1044-0305(93)85019-T

  70. 70.

    Kohler, M., Leary, J.A.: Lc/Ms/Ms of carbohydrates with Postcolumn addition of metal chlorides using a Triaxial electrospray probe. Anal. Chem. 67(19), 3501–3508 (1995). https://doi.org/10.1021/ac00115a019

  71. 71.

    Marginean, I., Kronewitter, S.R., Moore, R.J., Slysz, G.W., Monroe, M.E., Anderson, G., Tang, K.Q., Smith, R.D.: Improving N-glycan coverage using HPLC-MS with electrospray ionization at subambient pressure. Anal. Chem. 84(21), 9208–9213 (2012). https://doi.org/10.1021/ac301961u

  72. 72.

    Ikonomou, M.G., Blades, A.T., Kebarle, P.: Electrospray mass-spectrometry of methanol and water solutions suppression of electric-discharge with Sf6 gas. J Am Soc Mass Spectrom. 2(6), 497–505 (1991). https://doi.org/10.1016/1044-0305(91)80038-9

  73. 73.

    Kebarle, P.: A brief overview of the present status of the mechanisms involved in electrospray mass spectrometry. J Mass Spectrom. 35(7), 804–817 (2000). https://doi.org/10.1002/1096-9888(200007)35:7<804::Aid-Jms22>3.0.Co;2-Q

  74. 74.

    Kurokawa, T., Wuhrer, M., Lochnit, G., Geyer, H., Markl, J., Geyer, R.: Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with gal (β1–6) man-motifs. Eur. J. Biochem. 269(22), 5459–5473 (2002)

  75. 75.

    Schmidt, A., Karas, M., Dulcks, T.: Effect of different solution flow rates on analyte ion signals in nano-ESI MS, or: when does ESI turn into nano-ESI? J. Am. Soc. Mass Spectrom. 14(5), 492–500 (2003). https://doi.org/10.1016/S1044-0305(03)00128-4

  76. 76.

    Kebarle, P., Verkerk, U.H.: Electrospray: from ions in solution to ions in the gas phase, what we know now. Mass Spectrom. Rev. 28(6), 898–917 (2009). https://doi.org/10.1002/mas.20247

  77. 77.

    Yamashita, M., Fenn, J.B.: Negative-ion production with the electrospray ion-source. J. Phys. Chem. 88(20), 4671–4675 (1984). https://doi.org/10.1021/j150664a046

  78. 78.

    Yamashita, M., Fenn, J.B.: Electrospray ion-source - another variation on the free-jet theme. J. Phys. Chem. 88(20), 4451–4459 (1984). https://doi.org/10.1021/j150664a002

  79. 79.

    Staples, G.O., Naimy, H., Yin, H., Kileen, K., Kraiczek, K., Costello, C.E., Zaia, J.: Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow. Anal. Chem. 82(2), 516–522 (2010). https://doi.org/10.1021/ac901706f

  80. 80.

    Ni, W., Bones, J., Karger, B.L.: In-depth characterization of N-linked oligosaccharides using fluoride-mediated negative ion microfluidic chip LC-MS. Anal. Chem. 85(6), 3127–3135 (2013). https://doi.org/10.1021/ac3031898

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Acknowledgements

TNK and ER were supported by the European Commission (EC) under the FP7 project “HTP-GlycoMet – Methods for high-throughput glycoproteomic analysis” (grant no. 324400). ER acknowledges support by the German Research Foundation (DFG) under the project “The concert of dolichol-based glycosylation: from molecules to disease models” (grant identifier FOR2509), by the German Federal Ministry of Education and Research (BMBF) under the project “Die Golgi Glykan Fabrik 2.0” (grant identifier 031C557), and by the European Commission (EC) under the H2020 project “IMforFuture” (grant no. 721815).

We want to thank Marcus Hoffmann and Alexander Behne for their contribution in editing the manuscript.

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Correspondence to Erdmann Rapp.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Nguyen-Khuong, T., Pralow, A., Reichl, U. et al. Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow. Glycoconj J 35, 499–509 (2018). https://doi.org/10.1007/s10719-018-9848-1

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Keywords

  • Glycans
  • Glycomics
  • Mass spectrometry
  • Nano-electrospray
  • Nano-liquid chromatography
  • Negative ion mode
  • Orbitrap
  • Porous graphitized carbon
  • Post-column make-up flow
  • Stability