Comparison of Resins for Metal Oxide Affinity Chromatography with Mass Spectrometry Detection for the Determination of Phosphopeptides

Protein phosphorylation is a crucial regulatory mechanism in majority of biological processes. During MS, there is a general need to diminish suppression effect of non-phosphorylated peptides and counterbalance low abundance and insufficient ionization of phosphopeptides. Therefore, selective enrichment of their content in complex mixture has become an indispensable part of any phosphoproteomic study. In this work we employed metal oxide affinity chromatography (MOAC) approach. We have compared classic approach of mixing TiO2 and peptides in a microtube with microcolumns - commercial tips NuTips (TiO2/ZrO2 1:1) and TopTips (R) (TiO2, TiO2/ZrO2 1:1, and ZrO2). Selectivity of the given media towards phosphopeptides was tested on a tryptic digest of mixture of bovine proteins: /-casein and fetuin (phosphoproteins) with myoglobin and bovine serum albumin (non-phosphorylated proteins) in ratio 1:1:5:5 and 1:1:50:50, respectively. After enrichment, the obtained eluates were analyzed by tandem mass spectrometry (MALDI-TOF/TOF) on ABI 4800 in positive reflectron mode. To each media we applied four different protocols with different composition of loading and washing buffers and we compared efficiency of three displacers (1M lactic acid, 350mg/ml DHB, and 0.1M glutamic acid). In our settings, NuTips (R) proved as the most efficient media for analysis of low complex samples, since they exhibited the highest phosphoselectivity. Surprisingly, the Titansphere 5 mu m particles outperformed mixed TopTips, which against our expectations showed the lowest binding selectivity and reproducibility even after addition of three different displacers.