Selective Zirconium Dioxide-Based Enrichment of Phosphorylated Peptides for Mass Spectrometric Analysis

TLDR

Protein phosphorylation is dynamic and low in abundance, so enrichment of phosphopeptides is essential before mass spectrometry, and although TiO₂ columns have been used, they suffer from poor selectivity and reproducibility. The study introduces zirconium dioxide microtips as a novel phosphopeptide enrichment tool for mass spectrometric analysis. ZrO₂ microtips match TiO₂ in overall performance but preferentially isolate singly phosphorylated peptides, whereas TiO₂ favors multiply phosphorylated ones, making the two materials complementary; enzyme choice had no effect.

Abstract

Due to the dynamic nature and low stoichiometry of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Several phosphopeptide isolation strategies have been presented in the literature, including immobilized metal ion affinity chromatography. However, that technique suffers from poor selectivity and reproducibility. Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for phosphopeptide isolation prior to mass spectrometric analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two chromatographic materials possess complementary properties. For α- and β-casein, Glu-C digestion provided no evident advantage compared to trypsin digestion when combined with TiO2 or ZrO2 phosphopeptide enrichment.