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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

Abstract

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.

Keywords

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

Notes

Acknowledgements

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.

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© American Society for Mass Spectrometry 2019

Authors and Affiliations

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

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