Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays
Profiling Protein Tyrosine Phosphatase Specificity with Self-Assembled Monolayers for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Peptide Arrays
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DOI：
10.1021/acscombsci.9b00152
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发表时间：
2019-11-01
影响因子：
--
通讯作者：
Mrksich, Milan
中科院分区：
文献类型：
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作者：
Huang, Che-Fan;Mrksich, Milan
磷酸酶和激酶的相反活性决定了蛋白质的磷酸化状态，然而激酶在细胞过程的研究中受到了不成比例的关注，磷酸酶的作用仍然不太清楚。这篇研究文章描述了使用磷酸酪氨酸含有肽阵列与基质辅助激光解吸/电离（MALDI）质谱直接配置磷酸酶底物选择性。22个蛋白酪氨酸磷酸酶的特点与阵列，以提供其特异性的配置文件。数据分析显示，底物中的某些残基对所有测试酶的活性具有保守效应，包括在磷酸酪氨酸的任一侧包含碱性赖氨酸或精氨酸残基降低活性的一般规则。这一发现也为R1152 Q突变体在胰岛素受体中的作用提供了新的视角，已知R1152 Q突变体表现出较低的磷酸化水平，这项工作表明这可能是由于磷酸酶活性增加。使用自组装单分子膜基质辅助激光解吸/电离质谱（SAMDI-MS），以提供一个快速和定量测定磷酸酶将是重要的，以获得更全面的了解这一重要的酶类的生物化学和生物学。
The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This Research Article describes the use of phosphotyrosine-containing peptide arrays together with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to directly profile phosphatase substrate selectivities. Twenty-two protein tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of a R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level and which this work suggests may be due to an increased activity toward phosphatase enzymes. The use of self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.