Hwang Leekyoung, Ayaz-Guner Serife, Gregorich Zachery R, Cai Wenxuan, Valeja Santosh G, Jin Song, Ge Ying
Department of Chemistry, ‡Department of Cell and Regenerative Biology, §Molecular and Cellular Pharmacology Program, and ∥Human Proteomics Program, University of Wisconsin-Madison , Madison, Wisconsin 53719, United States.
J Am Chem Soc. 2015 Feb 25;137(7):2432-5. doi: 10.1021/ja511833y. Epub 2015 Feb 11.
Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.
由于磷酸化蛋白质丰度低以及磷酸化的化学计量比低，蛋白质磷酸化分析仍然是一项重大挑战，这就需要对磷酸化蛋白质进行有效的富集。在此，我们开发了超顺磁性纳米颗粒（NPs），其表面由多价配体分子功能化，这些配体分子能特异性结合任何磷酸化蛋白质上的磷酸基团。如通过使用磷酸化蛋白质特异性染色的SDS-PAGE分析以及对富集的磷酸化蛋白质进行质谱分析所证实的，这些纳米颗粒能以高特异性从复杂的细胞和组织裂解物中富集磷酸化蛋白质。该方法能够实现对完整磷酸化蛋白质的通用且有效的捕获、富集和检测，从而对磷酸化蛋白质组进行全面分析。
