期刊
JOURNAL OF PROTEOME RESEARCH
卷 7, 期 3, 页码 1078-1087出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr700655d
关键词
enrichment; Fe3+-immobilized magnetic nanoparticles; mass spectrometry; phosphorylation; plasma membrane
Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe3+- immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein a-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDI-TOFMS analysis. The lowest detectable concentration is 5 x 10(-11) M for 100 mu L of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation Sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据