Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction

Identification of phosphopeptides by MS is challenging due to their relatively low abundance in proteomic samples and their limited ionization efficiency. Various affinity enrichment methods have been used in the literature. Titanium dioxide SPE devices have been recently proposed as in alternative to immobilized metal affinity chromatography for phosphopeptide enrichment. This study evaluates the TiO2 method using sorbent packed in a 96 well microscale extraction plate operated using a vacuum manifold. The phosphopeptide recovery and enrichment selectivity were investigated at various loading conditions. The effectiveness of organic additives such as dihydroxybenzoic acid derivatives and other nonaliphalic carboxylic acids on enrichment selectivity wits examined. The performance of TiO2 was compared to IMAC sorbent. The results suggest that various additives improve the enrichment selectivity by effectively interfering with the acidic peptides binding to TiO2 sorbent. Interaction of phosphopeptides with sorbent is also affected, which leads to overall reduction in phosphopeptide recovery. The new SPE device was successfully utilized for the extraction of phosphopeptides from Yeast lysate digest using 2.5-dihydroxybenzoic acid to minimize the interference from nonphosphorylated peptides.