Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
Analyst. 2011 Nov 7;136(21):4454-9. doi: 10.1039/c1an15334j. Epub 2011 Sep 7.
Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(II,III) oxide (magnetite, Fe(3)O(4)) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis.
几种方法可用于提高磷酸化蛋白质的富集程度。在本文中，我们使用磁性氧化铁(II,III)纳米粒子（磁铁矿，Fe3O4）在放射状微结构硅芯片上对磷酸肽进行了富集，然后使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）进行分析，无需进一步的纯化过程。我们已经开发出一种放射状微结构芯片，可用于 MALDI-TOFMS 分析的样品浓缩。将磷酸化蛋白消化物和磁性氧化铁纳米粒子水溶液沉积在放射状微结构硅芯片的中央区域，实现了磷酸肽的芯片上富集。显微镜分析证实，应用的样品被限制在中央区域。聚焦在芯片上的样品斑点比具有相同总体积的未修饰板上的斑点小得多。使用了不同的添加剂并优化了工艺，以最小化非磷酸肽的干扰。这些数据共同表明，我们的芯片上磷酸肽富集方案是一种快速、易用的磷酸蛋白质组分析方法。
