A Multidimensional System for Phosphopeptide Analysis Using TiO2 Enrichment and Ion-exchange Chromatography with Mass Spectrometry

Although offline enrichment of phosphorylated peptides is widely used, enrichment for phosphopeptides using TiO2 is often performed manually, which is labor-intensive and can lead to irreproducible results. To address the problems associated with offline enrichment and to improve the effectiveness of phosphopeptide detection, we developed an automated online enrichment system for phosphopeptide analysis. A standard protein mixture comprising BSA, fetuin, crystalline, α-casein and β-casein, and ovalbumin was assessed using our new system. Our multidimensional system has four main parts: a sample pump, a 20-mm TiO2-based column, a weak anion-exchange, and a strong cation-exchange (2:1 WAX:SCX) separation column with LC/MS. Phosphorylated peptides were successfully detected using the TiO2-based online system with little interference from nonphosphorylated peptides. Our results confirmed that our online enrichment system is a simple and efficient method for detecting phosphorylated peptides