Layer-by-layer assembled magnetic bimetallic metal-organic framework composite for global phosphopeptide enrichment

The highly selective enrichment of phosphopeptides is vital for the research of phosphorylated proteomics. Herein, a magnetic bimetallic metal-organic framework (MOF) with Zr-O and Ti-O clusters (denoted as Fe₃O₄@SiO₂@(Zr-Ti-PTA)₁₅) has been fabricated using a layer-by-layer assembly approach via the coordination of metal ions with carboxyl groups. The composite has large specific surface area (135.84 m² g^-1) and superparamagnetism (62.03 emu g^-1). Compared with single-metal cluster MOF, the Fe₃O₄@SiO₂@(Zr-Ti-PTA)₁₅ showed higher binding capacity (100 mg g^-1), enhanced affinity, better selectivity (n_β-casein : n_BSA = 1 : 2000) and sensitivity (0.3 fmol/μL) towards phosphopeptides due to the combination of the dual metal clusters. Furthermore, the Fe₃O₄@SiO₂@ (Zr-Ti-PTA)₁₅ was successfully applied to extracting phosphopeptides from non-fat milk tryptic digest, and 10 mono-phosphopeptides and 22 multi-phosphopeptides were captured. As a result, 1294 phosphopeptides (476 multi-phosphopeptides and 818 mono-phosphopeptides) were selectively extracted and detected from tryptic digest of rat brain extract, indicating its great application potential in phosphorylated proteomics research.