Highly specific phosphopeptide enrichment by titanium(IV) cross-linked chitosan composite

• High sensitive and selective phosphopeptides enrichment by titanium(IV) cross-linked chitosan composite (Ti-CTS).
• Natural chitosan is applied for IMAC supporting material.
• Phosphopeptides in standard proteins (casein variants) and two real samples (non-fat milk and serum) were well enriched by Ti-CTS.

Natural chitosan was applied as supporting material for Ti(IV) based immobilized metal ion affinity chromatographic (IMAC) material (Ti-CTS). Compared with other polymer based IMAC, Ti-CTS can save the cockamamie synthesis procedures and be easy to obtain. The morphology, surface area, pore volume and elemental composition of Ti-CTS were revealed by scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) method and X-ray photoelectron spectroscopy (XPS). Tryptic digest products from several standard proteins and two real samples (non-fat milk and serum) were enriched using Ti-CTS to demonstrate the efficiency of this method. The results showed that this composite enables high sensitive and selective phosphopeptide enrichment from casein variants, non-fat milk and human serum. Furthermore, multi-phosphorylated peptides with three serine phospholated sites (S*S*S*) demonstrated high affinity to Ti-CTS. Hence, this method had great potential for future studies of complex phosphoproteomes and especially multi-phosphorylated peptides.