Preparation of monodisperse immobilized Ti(4+) affinity chromatography microspheres for specific enrichment of...

This study presented an approach to prepare monodisperse immobilized Ti(4+) affinity chromatography (Ti(4+)-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8mum were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13mum were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti(4+) ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti(4+)-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of alpha-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5fmol for standard tryptic digest of beta-casein. The Ti(4+)-IMAC microspheres were compared with commercial Fe(3+)-IMAC adsorbent and homemade Zr(4+)-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe(3+)-IMAC, Zr(4+)-IMAC and Ti(4+)-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti(4+)-IMAC had the best performance for enrichment of phosphopeptides.