Organic-Inorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column for analysis of mitochondrial phosphoproteome

A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic-inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 μm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti ⁴⁺ chelation. By such a hybrid silica monolithic Ti⁴⁺-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of α-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti⁴⁺-IMAC monolithic column were measured to be 1.4 μmol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti⁴⁺-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.