title: Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides.
abstract: 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 Ga(3+) chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga(3+)-ATP-MNPs). The high specificity of Ga(3+)-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. Ga(3+)-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 Ga(3+)-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 Ga(3+)-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 Ga(3+). Moreover, the unique magnetic core of Ga(3+)-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.
authors.affiliation: Liaoning Provincial Key Laboratory for Proteomics, Dalian Medical University, Dalian 116044, China.
authors.forename: Liyuan
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: lihuazhang@dicp.ac.cn.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Liaoning Provincial Key Laboratory for Proteomics, Dalian Medical University, Dalian 116044, China.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Shao
mesh_terms: D000241:Adenosine / Q000737:chemistry*; D005708:Gallium / Q000737:chemistry*; D057230:Limit of Detection; D008280:Magnetics; D008854:Microscopy, Electron; D010748:Phosphopeptides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D005708:Gallium; D000241:Adenosine
keywords: Adenosine triphosphate; Gallium ion; Immobilized metal ion affinity chromatography; Magnetic nanoparticles; Multi-phosphopeptide