COMPARISON OF RESINS FOR METAL OXIDE-AFFINITY CHROMATOGRAPHY WITH MASS SPECTROMETRY DETECTION FOR THE DETERMINATION OF PHOSPHOPEPTIDES

摘要

We have compared several different media for metal oxide affinity chromatography (MOAC) - a popular approach for enrichment of phosphopeptides prior MS analysis: TiO_2 particles Titansphere used in a microtube and commercial tips NuTips (TiO_2/ZrO_2 1:1) and TopTips (TiO_2, TiO_2/ZrO_2 1:1, and ZrO_2). Titansphere TiO_2 chromatography and NuTips with 0.1 M glutamic acid (protocol #4) to have the highest enrichment efficiency within all protocols tested. However, NuTips outperformed the classical Titansphere approach in total number of phosphopeptides detected in MALDI-TOF MS. Titanspher in our hands proved as the most efficient media for analysis of high complex samples, while NuTips proved as the most efficient media for analysis of low complex samples. Against our expectations, all 3 types of TopTips tested in our experiments exhibited lower binding selectivity and reproducibility than Titansphere particles and NuTips columns despite extensive washing with loading and washing solutions and addition of three different displacers for acidic peptides (which in general increased the phosphoselectivity of TopTips notably). Titansphere will be used for our phosphoproteomic study of human leukaemic cells after irradiation.