title: Highly efficient enrichment of phosphopeptides by magnetic nanoparticles coated with zirconium phosphonate for phosphoproteome analysis.
abstract: The location of phosphorylation plays a vital role for the elucidation of biological processes. The challenge of low stoichiometry of phosphoproteins and signal suppression of phosphopeptides by nonphosphopeptides in mass spectrometry (MS) analysis makes the selective enrichment of phosphopeptides prior to MS analysis necessary. Besides the immobilized metal affinity chromatography (IMAC) method, some affinity methods based on nanoparticles displayed a higher enrichment efficiency for phosphopeptides such as Fe(3)O(4)/TiO2 and Fe(3)O(4)/ZrO(2) nanoparticles. To further improve the selectivity and compatibility of the affinity methods, a novel strategy based on magnetic nanoparticles coated with zirconium phosphonate for the enrichment of phosphopeptides has been developed in this study. Under optimized experimental conditions, 1 x 10(-9) M phosphopeptides in 50 microL tryptic digest of beta-casein could be enriched and identified successfully. Reliable results were also obtained for 1 x 10(-8) M phosphopeptides in 50 microL tryptic digest of beta-casein in the presence of nonphosphopeptides from a tryptic digest of bovine serum albumin (BSA) over 20 times in concentration. The performance of nanoparticles for use in a real sample was further demonstrated by employing the strong cation-exchange chromatography (SCX) fraction of a tryptic digest of a protein extract from Chang liver cells as a model sample. Experimental results show that the nanoparticles can be easily and effectively used for enrichment of phosphopeptides in low concentration. Most importantly, our approach is more compatible with commonly used SCX strategies than Fe(3+)-IMAC. The proposed method thus has great potential for future studies of large-scale phosphoproteomes.
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, No.33 Life Science Park Road, Changping District, Beijing 102206, PR China.
authors.forename: Junying
authors.initials: J
authors.lastname: Wei
authors.forename: Yangjun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Tan
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Liu
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
mesh_terms: D002460:Cell Line; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008280:Magnetics; D013058:Mass Spectrometry / Q000379:methods; D015336:Molecular Probe Techniques; D053758:Nanoparticles / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D020543:Proteome / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D015040:Zirconium / Q000737:chemistry*
chemical_list: D063065:Organophosphonates; D010748:Phosphopeptides; D020543:Proteome; D015040:Zirconium