Counterion Optimization Dramatically Improves Selectivity for Phosphopeptides and Glycopeptides in Electrostatic...

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.