Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides

  • Yusi Cui
  • Ka Yang
  • Dylan Nicholas Tabang
  • Junfeng Huang
  • Weiping Tang
  • Lingjun LiEmail author
Focus: Protein Post-translational Modifications: Research Article


Simultaneous enrichment of glyco- and phosphopeptides will benefit the studies of biological processes regulated by these posttranslational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom-designed multifunctional solid phase extraction (SPE) materials, operating strong anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH, and salt on the retention of glycosylated and phosphorylated peptides was evaluated. This study demonstrated good enrichment of glyco- and phosphopeptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phosphopeptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.


Posttranslational modifications (PTMs) Strong anion exchange (SAX) Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) Glycosylation Phosphorylation Enrichment 



We would like to acknowledge Dr. Andrew Alpert from PolyLC Inc. for helpful discussions. This research was supported in part by the National Institutes of Health grants U01CA231081, R01 DK071801, R21 AG055377, and RF1 AG052324 (to LL). The Orbitrap instruments were purchased through the support of an NIH shared instrument grant (NIH-NCRR S10RR029531 to LL) and Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison. LL acknowledges a Vilas Distinguished Achievement Professorship and a Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin-Madison School of Pharmacy.


  1. 1.
    Ke, M., Shen, H., Wang, L., Luo, S., Lin, L., Yang, J., Tian, R.: Identification, quantification, and site localization of protein posttranslational modifications via mass spectrometry-based proteomics. In: Mirzaei, H., Carrasco, M. (eds.) Modern proteomics—sample preparation, analysis and practical applications, pp. 345–382. Springer International Publishing, Cham (2016)Google Scholar
  2. 2.
    Cohen, P.: The origins of protein phosphorylation. Nat. Cell Biol. 4, E127–E130 (2002)Google Scholar
  3. 3.
    Apweiler, R., Hermjakob, H., Sharon, N.: On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta, Gen. Subj. 1473, 4–8 (1999)Google Scholar
  4. 4.
    Moremen, K.W., Tiemeyer, M., Nairn, A.V.: Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462 (2012)Google Scholar
  5. 5.
    Lee, R.T., Lauc, G., Lee, Y.C.: Glycoproteomics: protein modifications for versatile functions. EMBO Rep. 6, 1018–1022 (2005)Google Scholar
  6. 6.
    Ihara, Y., Nukina, N., Miura, R., Ogawara, M.: Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer’s disease. J. Biochem. 99, 1807–1810 (1986)Google Scholar
  7. 7.
    Maverakis, E., Kim, K., Shimoda, M., Gershwin, M.E., Patel, F., Wilken, R., Raychaudhuri, S., Ruhaak, L.R., Lebrilla, C.B.: Glycans in the immune system and the altered glycan theory of autoimmunity: a critical review. J. Autoimmun. 57, 1–13 (2015)Google Scholar
  8. 8.
    Hakomori, S.: Glycosylation defining cancer malignancy: new wine in an old bottle. Proc. Natl. Acad. Sci. U. S. A. 99, 10231–10233 (2002)Google Scholar
  9. 9.
    Schedin-Weiss, S., Winblad, B., Tjernberg, L.O.: The role of protein glycosylation in Alzheimer disease. FEBS J. 281, 46–62 (2014)Google Scholar
  10. 10.
    Lassen, P.S., Thygesen, C., Larsen, M.R., Kempf, S.J.: Understanding Alzheimer’s disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles: a chance for new biomarkers in neuroproteomics? J. Proteome. 161, 11–25 (2017)Google Scholar
  11. 11.
    Karlsson, H.K.R., Zierath, J.R., Kane, S., Krook, A., Lienhard, G.E., Wallberg-Henriksson, H.: Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects. Diabetes. 54, 1692–1697 (2005)Google Scholar
  12. 12.
    Boucher, J., Kleinridders, A., Kahn, C.R.: Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb. Perspect. Biol. 6, a009191Google Scholar
  13. 13.
    Freeze, H.H., Eklund, E.A., Ng, B.G., Patterson, M.C.: Neurology of inherited glycosylation disorders. Lancet Neurol. 11, 453–466 (2012)Google Scholar
  14. 14.
    Kanninen, K., Goldsteins, G., Auriola, S., Alafuzoff, I., Koistinaho, J.: Glycosylation changes in Alzheimer’s disease as revealed by a proteomic approach. Neurosci. Lett. 367, 235–240 (2004)Google Scholar
  15. 15.
    Wang, X., Li, D., Wu, G., Bazer, F.W.: Functional roles of fructose: crosstalk between O-linked glycosylation and phosphorylation of Akt-TSC2-mtor cell signaling cascade in ovine trophectoderm cells. Biol. Reprod. 95, 102 (2016) 1–17Google Scholar
  16. 16.
    Hang, Q., Isaji, T., Hou, S., Im, S., Fukuda, T., Gu, J.: Integrinα5 suppresses the phosphorylation of epidermal growth factor receptor and its cellular signaling of cell proliferation via N-glycosylation. J. Biol. Chem. 290, 29345–29360 (2015)Google Scholar
  17. 17.
    Aebersold, R., Mann, M.: Mass spectrometry-based proteomics. Nature. 422, 198–207 (2003)Google Scholar
  18. 18.
    Domon, B., Aebersold, R.: Mass spectrometry and protein analysis. Science. 312, 212–217 (2006)Google Scholar
  19. 19.
    Yates, J.R., Ruse, C.I., Nakorchevsky, A.: Proteomics by mass spectrometry: approaches, advances, and applications. Annu. Rev. Biomed. Eng. 11, 49–79 (2009)Google Scholar
  20. 20.
    Bekker-Jensen, D.B., Kelstrup, C.D., Batth, T.S., Larsen, S.C., Haldrup, C., Bramsen, J.B., Sørensen, K.D., Høyer, S., Ørntoft, T.F., Andersen, C.L., Nielsen, M.L., Olsen, J.V.: An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst. 4, 587–599.e584 (2017)Google Scholar
  21. 21.
    Riley, N.M., Hebert, A.S., Coon, J.J.: Proteomics moves into the fast lane. Cell Syst. 2, 142–143 (2016)Google Scholar
  22. 22.
    Hebert, A.S., Richards, A.L., Bailey, D.J., Ulbrich, A., Coughlin, E.E., Westphall, M.S., Coon, J.J.: The one hour yeast proteome. Mol. Cell. Proteomics. 13, 339–347 (2014)Google Scholar
  23. 23.
    Schroeder, M.J., Shabanowitz, J., Schwartz, J.C., Hunt, D.F., Coon, J.J.: A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. Anal. Chem. 76, 3590–3598 (2004)Google Scholar
  24. 24.
    Syka, J.E.P., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101, 9528–9533 (2004)Google Scholar
  25. 25.
    Zubarev, R.A.: Electron-capture dissociation tandem mass spectrometry. Curr. Opin. Biotechnol. 15, 12–16 (2004)Google Scholar
  26. 26.
    Reiding, K.R., Bondt, A., Franc, V., Heck, A.J.R.: The benefits of hybrid fragmentation methods for glycoproteomics. Trends Anal. Chem. 108, 260–268 (2018)Google Scholar
  27. 27.
    Riley, N.M., Hebert, A.S., Dürnberger, G., Stanek, F., Mechtler, K., Westphall, M.S., Coon, J.J.: Phosphoproteomics with activated ion electron transfer dissociation. Anal. Chem. 89, 6367–6376 (2017)Google Scholar
  28. 28.
    Glover, M.S., Yu, Q., Chen, Z., Shi, X., Kent, K.C., Li, L.: Characterization of intact sialylated glycopeptides and phosphorylated glycopeptides from IMAC enriched samples by EThcD fragmentation: toward combining phosphoproteomics and glycoproteomics. Int. J. Mass Spectrom. 427, 35–42 (2018)Google Scholar
  29. 29.
    Yang, Y., Liu, F., Franc, V., Halim, L.A., Schellekens, H., Heck, A.J.R.: Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 7, 13397: 1–13397:10 (2016)Google Scholar
  30. 30.
    Yu, Q., Shi, X., Feng, Y., Kent, K.C., Li, L.: Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies. Anal. Chim. Acta. 968, 40–49 (2017)Google Scholar
  31. 31.
    Mommen, G.P.M., Frese, C.K., Meiring, H.D., van Gaans-van den Brink, J., de Jong, A.P.J.M., van Els, C.A.C.M., Heck, A.J.R.: Expanding the detectable HLA peptide repertoire using electron-transfer/higher-energy collision dissociation (EThcD). Proc. Natl. Acad. Sci. U. S. A. 111, 4507–4512 (2014)Google Scholar
  32. 32.
    Yu, Q., Wang, B., Chen, Z., Urabe, G., Glover, M.S., Shi, X., Guo, L.-W., Kent, K.C., Li, L.: Electron-transfer/higher-energy collision dissociation (EThcD)-enabled intact glycopeptide/glycoproteome characterization. J. Am. Soc. Mass Spectrom. 28, 1751–1764 (2017)Google Scholar
  33. 33.
    Yu, Q., Canales, A., Glover, M.S., Das, R., Shi, X., Liu, Y., Keller, M.P., Attie, A.D., Li, L.: Targeted mass spectrometry approach enabled discovery of O-glycosylated insulin and related signaling peptides in mouse and human pancreatic islets. Anal. Chem. 89, 9184–9191 (2017)Google Scholar
  34. 34.
    Diedrich, J.K., Pinto, A.F.M., Yates III, J.R.: Energy dependence of HCD on peptide fragmentation: stepped collisional energy finds the sweet spot. J. Am. Soc. Mass Spectrom. 24, 1690–1699 (2013)Google Scholar
  35. 35.
    Yang, H., Yang, C., Sun, T.: Characterization of glycopeptides using a stepped higher-energy C-trap dissociation approach on a hybrid quadrupole orbitrap. Rapid Commun. Mass Spectrom. 32, 1353–1362 (2018)Google Scholar
  36. 36.
    Palmisano, G., Lendal, S.E., Engholm-Keller, K., Leth-Larsen, R., Parker, B.L., Larsen, M.R.: Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry. Nat. Protoc. 5, 1974–1982 (2010)Google Scholar
  37. 37.
    Kweon, H.K., Håkansson, K.: Selective zirconium dioxide-based enrichment of phosphorylated peptides for mass spectrometric analysis. Anal. Chem. 78, 1743–1749 (2006)Google Scholar
  38. 38.
    Feng, S., Ye, M., Zhou, H., Jiang, X., Jiang, X., Zou, H., Gong, B.: Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis. Mol. Cell. Proteomics. 6, 1656–1665 (2007)Google Scholar
  39. 39.
    Zhou, H., Ye, M., Dong, J., Corradini, E., Cristobal, A., Heck, A.J.R., Zou, H., Mohammed, S.: Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography. Nat. Protoc. 8, 461–480 (2013)Google Scholar
  40. 40.
    Hong, Y., Yao, Y., Zhao, H., Sheng, Q., Ye, M., Yu, C., Lan, M.: Dendritic mesoporous silica nanoparticles with abundant Ti4+ for phosphopeptide enrichment from cancer cells with 96% specificity. Anal. Chem. 90, 7617–7625 (2018)Google Scholar
  41. 41.
    Iliuk, A.B., Martin, V.A., Alicie, B.M., Geahlen, R.L., Tao, W.A.: In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion functionalized soluble nanopolymers. Mol. Cell. Proteomics. 9, 2162–2172 (2010)Google Scholar
  42. 42.
    Tao, W.A., Wollscheid, B., O'Brien, R., Eng, J.K., Li, X.-j., Bodenmiller, B., Watts, J.D., Hood, L., Aebersold, R.: Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat. Methods. 2, 591–598 (2005)Google Scholar
  43. 43.
    Zhu, F., Trinidad, J.C., Clemmer, D.E.: Glycopeptide site heterogeneity and structural diversity determined by combined lectin affinity chromatography/IMS/CID/MS techniques. J. Am. Soc. Mass Spectrom. 26, 1092–1102 (2015)Google Scholar
  44. 44.
    Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M., Hirabayashi, J., Kasai, K.-i., Takahashi, N., Isobe, T.: Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nat. Biotechnol. 21, 667–672 (2003)Google Scholar
  45. 45.
    Chen, R., Jiang, X., Sun, D., Han, G., Wang, F., Ye, M., Wang, L., Zou, H.: Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. J. Proteome Res. 8, 651–661 (2009)Google Scholar
  46. 46.
    Zhang, H., Li, X.-j., Martin, D.B., Aebersold, R.: Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666 (2003)Google Scholar
  47. 47.
    Tian, Y., Zhou, Y., Elliott, S., Aebersold, R., Zhang, H.: Solid-phase extraction of N-linked glycopeptides. Nat. Protoc. 2, 334–339 (2007)Google Scholar
  48. 48.
    Zhou, Y., Aebersold, R., Zhang, H.: Isolation of N-linked glycopeptides from plasma. Anal. Chem. 79, 5826–5837 (2007)Google Scholar
  49. 49.
    Chen, W., Smeekens, J.M., Wu, R.: A universal chemical enrichment method for mapping the yeast N-glycoproteome by MS. Mol. Cell. Proteomics. 13, 1563–1572 (2014)Google Scholar
  50. 50.
    Xiao, H., Chen, W., Smeekens, J.M., Wu, R.: An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins. Nat. Commun. 9, 1692: 1–1692:12 (2018)Google Scholar
  51. 51.
    Xiao, H., Wu, R.: Simultaneous quantitation of glycoprotein degradation and synthesis rates by integrating isotope labeling, chemical enrichment, and multiplexed proteomics. Anal. Chem. 89, 10361–10367 (2017)Google Scholar
  52. 52.
    Mysling, S., Palmisano, G., Højrup, P., Thaysen-Andersen, M.: Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. Anal. Chem. 82, 5598–5609 (2010)Google Scholar
  53. 53.
    Hägglund, P., Bunkenborg, J., Elortza, F., Jensen, O.N., Roepstorff, P.: A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J. Proteome Res. 3, 556–566 (2004)Google Scholar
  54. 54.
    Calvano, C.D., Zambonin, C.G., Jensen, O.N.: Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry. J. Proteome. 71, 304–317 (2008)Google Scholar
  55. 55.
    Yu, L., Li, X., Guo, Z., Zhang, X., Liang, X.: Hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage. Chem. Eur. J. 15, 12618–12626 (2009)Google Scholar
  56. 56.
    Xu, D., Yan, G., Gao, M., Deng, C., Zhang, X.: Highly selective SiO2–NH2@TiO2 hollow microspheres for simultaneous enrichment of phosphopeptides and glycopeptides. Anal. Bioanal. Chem. 409, 1607–1614 (2017)Google Scholar
  57. 57.
    Zou, X., Jie, J., Yang, B.: Single-step enrichment of N-glycopeptides and phosphopeptides with novel multifunctional Ti4+-immobilized dendritic polyglycerol coated chitosan nanomaterials. Anal. Chem. 89, 7520–7526 (2017)Google Scholar
  58. 58.
    Hong, Y., Zhao, H., Pu, C., Zhan, Q., Sheng, Q., Lan, M.: Hydrophilic phytic acid-coated magnetic graphene for titanium(IV) immobilization as a novel hydrophilic interaction liquid chromatography-immobilized metal affinity chromatography platform for glyco- and phosphopeptide enrichment with controllable selectivity. Anal. Chem. 90, 11008–11015 (2018)Google Scholar
  59. 59.
    Di Palma, S., Hennrich, M.L., Heck, A.J.R., Mohammed, S.: Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis. J. Proteome. 75, 3791–3813 (2012)Google Scholar
  60. 60.
    Taouatas, N., Altelaar, A.F.M., Drugan, M.M., Helbig, A.O., Mohammed, S., Heck, A.J.R.: Strong cation exchange-based fractionation of Lys-N-generated peptides facilitates the targeted analysis of post-translational modifications. Mol. Cell. Proteomics. 8, 190–200 (2009)Google Scholar
  61. 61.
    Hennrich, M.L., Groenewold, V., Kops, G.J.P.L., Heck, A.J.R., Mohammed, S.: Improving depth in phosphoproteomics by using a strong cation exchange-weak anion exchange-reversed phase multidimensional separation approach. Anal. Chem. 83, 7137–7143 (2011)Google Scholar
  62. 62.
    Mohammed, S., Heck, A.J.R.: Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modifications. Curr. Opin. Biotechnol. 22, 9–16 (2011)Google Scholar
  63. 63.
    Alpert, A.J.: Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal. Chem. 80, 62–76 (2008)Google Scholar
  64. 64.
    Alpert, A.J., Hudecz, O., Mechtler, K.: Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography. Anal. Chem. 87, 4704–4711 (2015)Google Scholar
  65. 65.
    Totten, S.M., Feasley, C.L., Bermudez, A., Pitteri, S.J.: Parallel comparison of N-linked glycopeptide enrichment techniques reveals extensive glycoproteomic analysis of plasma enabled by SAX-ERLIC. J. Proteome Res. 16, 1249–1260 (2017)Google Scholar
  66. 66.
    Yang, W., Shah, P., Hu, Y., Toghi Eshghi, S., Sun, S., Liu, Y., Zhang, H.: Comparison of enrichment methods for intact N- and O-linked glycopeptides using strong anion exchange and hydrophilic interaction liquid chromatography. Anal. Chem. 89, 11193–11197 (2017)Google Scholar
  67. 67.
    Tran, T.H., Hwang, I.-J., Park, J.-M., Kim, J.-B., Lee, H.-K.: An application of electrostatic repulsion hydrophilic interaction chromatography in phospho- and glycoproteome profiling of epicardial adipose tissue in obesity mouse. Mass Spectrom. Lett. 3, 39–42 (2012)Google Scholar
  68. 68.
    Zhang, H., Guo, T., Li, X., Datta, A., Park, J.E., Yang, J., Lim, S.K., Tam, J.P., Sze, S.K.: Simultaneous characterization of glyco- and phosphoproteomes of mouse brain membrane proteome with electrostatic repulsion hydrophilic interaction chromatography. Mol. Cell. Proteomics. 9, 635–647 (2010)Google Scholar
  69. 69.
    Hao, P., Guo, T., Sze, S.K.: Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography. PLoS One. 6, e16844 (2011)Google Scholar
  70. 70.
    Ge, F., Tao, S., Bi, L., Zhang, Z., Zhang, X.E.: Proteomics: addressing the challenges of multiple myeloma. Acta Biochim. Biophys. Sin. 43, 89–95 (2011)Google Scholar
  71. 71.
    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, 5784–5793 (2014)Google Scholar
  72. 72.
    Selman, M.H.J., Hemayatkar, M., Deelder, A.M., Wuhrer, M.: Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides. Anal. Chem. 83, 2492–2499 (2011)Google Scholar
  73. 73.
    Darula, Z., Sherman, J., Medzihradszky, K.F.: How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides. Mol. Cell. Proteomics. 11, 1–10 (2012)Google Scholar
  74. 74.
    Alpert, A.J.: Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. A. 499, 177–196 (1990)Google Scholar
  75. 75.
    Hemström, P., Irgum, K.: Hydrophilic interaction chromatography. J. Sep. Sci. 29, 1784–1821 (2006)Google Scholar
  76. 76.
    Thannhauser, T.W., Shen, M., Sherwood, R., Howe, K., Fish, T., Yang, Y., Chen, W., Zhang, S.: A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry. Electrophoresis. 34, 2417–2431 (2013)Google Scholar
  77. 77.
    Ding, W., Hill, J.J., Kelly, J.: Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography. Anal. Chem. 79, 8891–8899 (2007)Google Scholar
  78. 78.
    Ding, W., Nothaft, H., Szymanski, C.M., Kelly, J.: Identification and quantification of glycoproteins using ion-pairing normal-phase liquid chromatography and mass spectrometry. Mol. Cell. Proteomics. 8, 2170–2185 (2009)Google Scholar
  79. 79.
    Wimley, W.C., Gawrisch, K., Creamer, T.P., White, S.H.: Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability. PNAS. 93, 2985 (1996)Google Scholar
  80. 80.
    Alpert, A.J.: Effect of salts on retention in hydrophilic interaction chromatography. J. Chromatogr. A. 1538, 45–53 (2018)Google Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WisconsinMadisonUSA
  2. 2.School of PharmacyUniversity of WisconsinMadisonUSA

Personalised recommendations