Sun Luo, Ghosh Inca, Barshevsky Tanya, Kochinyan Samvel, Xu Ming-Qun
New England Biolabs, Inc. Ipswich, MA 01938-2723, USA.
Methods. 2007 Jul;42(3):220-6. doi: 10.1016/j.ymeth.2007.02.012.
The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on solid supports.
蛋白质磷酸酶（PPs）底物特异性的研究极具挑战性，因为制备合适的磷酸化底物很困难。由蛋白激酶磷酸化的磷蛋白或化学合成的磷酸肽是PPs常用的底物。这两种类型的底物都有其优点和局限性。磷蛋白更接近生理相关的PP底物，但其制备技术要求高。合成磷酸肽比蛋白质有优势，因为它们可以很容易大量生产，并且其氨基酸序列可以设计成包含底物特异性的潜在决定因素。然而，与体内PP底物相比，短肽不太理想，并且通常与不同基质的结合较差且变化不定，导致在固体支持物上分析PP活性时灵敏度较低。在这项工作中，我们利用内含肽介导的蛋白质连接（IPL）技术来生成PPs的底物，将蛋白质和合成肽的优点结合在一个分子中。以一对一的化学计量比将合成磷酸肽与内含肽生成的载体蛋白（CP）连接，导致形成连接的磷蛋白（LPP）。采用三种广泛使用的检测方法，即斑点印迹阵列、蛋白质印迹和酶联免疫吸附测定（ELISA）来研究LPP底物上的PP活性。通过用磷酸特异性抗体检测剩余的磷酸化或未磷酸化情况来测量去磷酸化。数据显示在固体支持物检测中，LPP比游离肽具有优势。LPP与研究中使用的基质的结合增强，这显著提高了检测的灵敏度和一致性。此外，通过对检测样品进行系列稀释避免了信号饱和。本报告描述了制备LPP底物及其在基于固定在固体支持物上的PP检测中的详细实验步骤。
