Loss of Phosphate Groups during Phosphopeptide Enrichment with TiO_2 Columns-Systematic Studies by MALDI-ToF MS and MALDI-FT-ICR MS

摘要

Enrichment of phosphopeptides with TiO_2 has become a method of choice prior to mass spectrometric identification of protein phosphorylation sites [1]. However, in almost all literature reports the presence of "unspecifically" isolated, non-phosphorylated peptides in the eluted samples has been mentioned. In order to investigate why non-phosphorylated peptides are found in the eluates, we have developed a MALDI MS-based analysis procedure using commercially available TiO_2-loaded pipette tips. We tested the influence of additives (DHB, citric acid, methionine) during loading of samples by investigating the elution profiles from the affinity columns in detail. Nex t, several MALDI matrices (DHB, SA, THAP, etc.) were interrogated concerning phosphopeptide ion signal yields. Our results show that dephosphorylation takes place during sample elution from TiO_2-loaded columns (Fig. 1).