Monodisperse Ti-immobilized macroporous adsorbent resins with polymer brush for improved multi-phosphopeptides enrichment in milk.

Enrichment of phosphopeptides before mass spectrometry (MS) analysis is essential due to the limitations of low abundance and poor ionization efficiency in complex biological samples. Immobilized metal affinity chromatography (IMAC), especially titanium ion (Ti)-IMAC, has become a popular strategy for enrichment of phosphopeptides due to high selectivity and sensitivity. Conventional Ti-immobilized macroporous adsorption resin (MAR) fabricated by monolayer modification can preferentially capture mono-phosphopeptide over multi-phosphopeptides, which takes on more functions in the regulation of cell behaviors of organism. In this paper, a kind of monodisperse MAR microsphere with functional polymer brush (Ti-Brush@MAR) was prepared and modified via surface-initiated atom transfer radical polymerization (SI-ATRP). Compared with common Ti-MAR without polymer brush, Ti-Brush@MAR exhibited high enrichment specificity not only for mono-phosphopeptides but also for multi-phosphopeptides in β-casein or milk digest samples. As a result, a total of 93 unique phosphopeptides mapped to 18 phosphoproteins were identified from 5 μL milk, and the limit of detection is 10 fmol. It is expected that Ti-Brush@MAR would be utilized to enrich both multi-phosphopeptides and mono-phosphopeptides in additional biological or food samples.