Yuan Elena Tianfei, Ino Yoko, Kawaguchi Maho, Kimura Yayoi, Hirano Hisashi, Kinoshita-Kikuta Emiko, Kinoshita Eiji, Koike Tohru
Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Advanced Medical Research Center, Yokohama City University, Yokohama, Japan.
Electrophoresis. 2017 Oct;38(19):2447-2455. doi: 10.1002/elps.201700175. Epub 2017 Jul 10.
Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
磷酸化肽是磷酸化蛋白质组研究中具有吸引力的目标。在此，我们介绍一种简单便捷的微量移液器吸头方法，通过使用1,3-双（吡啶-2-基甲基氨基）丙-2-醇（Phos-tag）的磷酸结合锌（II）配合物来分离磷酸化和非磷酸化肽。制备了一个含有10 μL用Phos-tag部分功能化的琼脂糖珠的200 μL微量移液器吸头。磷酸亲和分离的所有步骤（结合、洗涤和洗脱）均使用中性pH值的水性缓冲液进行。每个样品的整个分离方案所需时间不到30分钟。通过三次独立的分离实验，随后进行质谱（MS）分析，我们从人胚肾细胞裂解物中鉴定出1649个非冗余磷酸化肽（每次MS分析的肽样品源自25 μg蛋白质）。在各自实验中，鉴定出的磷酸化肽与总肽的平均比例>90%，显示出高选择性。此外，通过基于每个肽的归一化丰度的散点图证实了三次重复分析之间的高度相关性，该归一化丰度由Progenesis QI中的无标记肽相对定量分析计算得出。因此，这种微量移液器吸头方法将优先用作现有工具的替代方法，用于可靠地富集磷酸化肽。
