Zinc Ion-immobilized Magnetic Microspheres for Enrichment and Identification of Multi-phosphorylated Peptides by Mass...

The selective isolation of phosphorylated peptides and subsequent analysis using mass spectrometry is important for understanding how protein kinase and phosphatase signals can precisely modulate the on/off states of signal transduction pathways. However, the isolation and detection of multi-phosphorylated peptides is still limited due to their distinct affinity to various materials and their poor ionization efficiency. Here, we report a highly efficient and selective enrichment of phosphorylated peptides using binuclear Zn²⁺-dipicolylamine complex-coated magnetic microspheres (ZnMMs). ZnMMs can utilize the rapid and selective isolation/enrichment of phosphorylated peptides and the subsequent mass spectrometric analysis, given the intrinsic magnetic property of magnetic microspheres and the highly selective binding ability of the binuclear Zn²⁺-dipicolylamine complex to phosphate groups. α-Casein and β-casein were chosen for a proof-of-concept demonstration. We contemplated that phosphopeptides were selectively isolated and enriched from both the tryptic digests of casein proteins and mixed samples with a high degree of sensitivity by facilitating ZnMMs. Especially, ZnMMs showed high efficiency with multi-phosphopeptides, which are in general difficult to be examined by mass analysis on account of their poor ionization efficiency. For the model protein α, β-casein mixture of the tryptic digest, 17 phosphopeptides were identified with ZnMMs and 82% of the enriched phosphopeptides were multi-phosphorylated peptides, indicating that ZnMMs have excellent enrichment efficiency and strong affinity towards multi-phosphorylated peptides.