Robust Titanium Phenolate-Modified Microspheres as Reusable Affinity Materials for Selectively Capturing Phosphopeptides from Complicated Biosamples

Immobilized metal affinity chromatography (IMAC) has been proven to be an effective strategy for enrichment of phosphopeptides. Although various materials are used as IMAC adsorbents, most of them are disposable, which is not consistent with the concept of sustainability. Herein, a novel Ti4+-immobilized microsphere was designed and simply prepared by employing a cheap macroporous adsorption resin (MAR) with epoxy groups as the matrix and 3,4,5-trihydroxybenzaldehyde containing a pyrogallol group as the chelator to immobilize titanium (Ti4+). The resulting Ti4+-MAR, as an IMAC sorbent, was reusable, owing to strong coordination interaction between the pyrogallol ligand and phosphopeptides. The Ti4+ could not be dissociated after eluting the captured phosphopeptides from sorbents using ammonium hydroxide. However, the phenomenon of dissociating Ti4+ is apt to occur on the common phosphate-based Ti4+-IMAC materials because ammonium hydroxide would break the chelation between Ti4+ and the phosphate ligand and cause Ti4+ to fall off the materials. This novel Ti4+-MAR microspheres still exhibited an excellent enrichment effect to phosphopeptides from a β-casein digest even after 10 cycles of reuse. A total of 3305 unique phosphopeptides mapped to 1464 phosphoproteins were unambiguously identified from a HeLa cell digest employing Ti4+-MAR microspheres as sorbents, which was superior to that of commercial SPE-Ti-IMAC material under the same conditions. As a result, these novel titanium phenolate-modified MAR microspheres exhibited excellent performance of enrichment, low-cost and especial reusability, and demonstrated a promising prospect for commercialization in the future.