Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic Repulsion-Hydrophilic Interaction Chromatography

Abstract

A well-hydrated counterion can selectively and dramatically increase retention of a charged analyte in hydrophilic interaction chromatography. The effect is enhanced if the column is charged, as in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). This combination was exploited in proteomics for the isolation of peptides with certain post-translational modifications (PTMs). The best salt additive examined was magnesium trifluoroacetate. The well-hydrated Mg +2 ion promoted retention of peptides with functional groups that retained negative charge at low pH, while the poorly hydrated trifluoroacetate counterion tuned down the retention due to the basic residues. The result was an enhancement in selectivity ranging from 6-to 66-fold. These conditions were applied to a tryptic digest of mouse cortex. Gradient elution produced fractions enriched in peptides with phosphate, mannose-6-phosphate, and N-and O-linked glycans. The numbers of such peptides identified either equaled or exceeded the numbers afforded by the best alternative methods. This method is a productive and convenient way to isolate peptides simultaneously that contain a number of different PTMs, facilitating study of proteins with "crosstalk" modifications. The fractions from the ERLIC column were desalted prior to C-18-reversed phase liquid chromatography−tandem mass spectrometry analysis. Between 47−100% of the peptides with more than one phosphate or sialyl residue or with a mannose-6 phosphate group were not retained by a C-18 cartridge but were retained by a cartridge of porous graphitic carbon. This finding implies that the abundance of such peptides may have been significantly underestimated in some past studies.

Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade

“…Meanwhile, the addition of well‐hydrated cations to the solvent can improve the selectivity to glycopeptides. For example, the addition of magnesium trifluoroacetate (Mg 2+ ) to the solvent will form a well‐hydrated ion pair with sialic acid residues, which can enhance the selectivity for N‐ or O‐linked sialylated glycopeptides by five‐ to sixfold at high pH [69].…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…The SAX‐ERLIC is simple to use and showed comparable results with traditional HILIC for the capture of N‐ glycopeptides and sialylated O‐ glycopeptides; however, it showed less efficiency for the capture of non‐sialylated O‐ glycopeptides. Cui and coworkers [98] used a counterion‐ERLIC for the enrichment of phosphopeptides and glycopeptides. In this approach, a magnesium ion (Mg +2 ) is used to improve the retention of glycopeptides and phosphopeptides by interacting with their negatively charged functional groups observed at low pH.…”

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

“…Efforts to maximize the PTM information obtained with MS workflows have gained traction in recent years. Using LC-MS, glycosylation is often studied with phosphorylation, as they are among the most common PTMs (Glover et al, 2018;Cho et al, 2019;Cui et al, 2019;Zhou et al, 2020;Cui et al, 2021;Huang et al, 2021). Both PTMs are implicated in cell signaling, and O-glycans may modify Ser and Thr, also two possible residues for phosphorylation.…”