Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides

• Ga3+-ATP-MNPs have distinct selectivity for multi-phosphopeptides.
• Ga3+-ATP-MNPs provide sensitivity for multi-phosphopeptides at 30 amol.
• Ga3+-ATP-MNPs show superior efficiency for multi-phosphopeptides isolation.
• Ga3+-ATP-MNPs show great promise for the large-scale phosphoproteome study.

Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga3+ chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga3+-ATP-MNPs). The high specificity of Ga3+-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga3+-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga3+-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga3+-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga3+. Moreover, the unique magnetic core of Ga3+-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.

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