Inhibitory Covalent Labeling and Clickable-Eu-Tagging-Based ICPMS: Measurement of pH-Dependent Absolute Activities of the Cathepsins in Hepatocyte Lysosomes
Inhibitory Covalent Labeling and Clickable-Eu-Tagging-Based ICPMS: Measurement of pH-Dependent Absolute Activities of the Cathepsins in Hepatocyte Lysosomes
复制标题
基于抑制性共价标记和可点击 EU 标记的 ICPMS：测量肝细胞溶酶体中组织蛋白酶的 pH 依赖性绝对活性
DOI：
10.1021/acs.analchem.9b01662
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发表时间：
2019
影响因子：
7.4
通讯作者：
Qiuquan Wang
中科院分区：
文献类型：
--
作者：
Caixia Ji;Yong Liang;Fuchun Ge;Limin Yang;Qiuquan Wang
我们报告了一种抑制性共价标记和可点击元素标记的策略，用于使用电感耦合等离子体质谱（ICPMS）测量细胞中蛋白酶的绝对活性。设计并合成了环氧琥珀酰-亮氨酸-酪氨酸-6-氨基己酸-赖氨酸-氨基-Boc-炔（环氧琥珀酰-LYK-炔）以实现半胱氨酸组织蛋白酶的不可逆标记，记录其瞬时活性。L和Y协助环氧琥珀酰-LYK-炔进入Cys 25的去质子化-S-，位于长组织蛋白酶活性结构域的底部。使用叠氮基-DOTA-Eu通过生物正交1：1铜催化的叠氮化物-炔-环加成点击反应进行定量Eu标记。Eu标记物可以绝对定量使用153 Eu-物种-非特异性-同位素-稀释ICPMS耦合HPLC，作为Eu标尺，并允许我们同时测量pH依赖的组织蛋白酶B，L和S的活性以及在肝癌C7721和癌旁C7701细胞的溶酶体微环境中的pH。只要设计和合成合适的标记分子和元素标签，我们相信这种串联标记和标签ICPMS方法可以应用于测量细胞中其他蛋白酶的活性，提供更准确的蛋白酶生物功能信息，从而实现精确的临床诊断。
We report an inhibitory covalent labeling and clickable-element-tagging strategy for measuring the absolute activity of a protease in cells using inductively coupled plasma mass spectrometry (ICPMS). Epoxysuccinyl-leucine-tyrosine-6-aminocaproic-lysine-amino-Boc-alkyne (epoxysuccinyl-LYK-alkyne) was designed and synthesized to achieve irreversibly labeling of the cysteine cathepsins, recording their momentary activities. L and Y assisted epoxysuccinyl-LYK-alkyne in accessing the deprotonated −S–of Cys25, located at the bottom of the long cathepsin active domain. Quantitative Eu-tagging was followed using azido-DOTA-Eu through a bioorthogonal 1:1 copper-catalyzed azide-alkyne-cycloaddition click reaction. The Eu tag could be absolutely quantified using153Eu-species-nonspecific-isotope-dilution ICPMS coupled with HPLC, serving as a Eu ruler and allowing us to simultaneously measure the pH-dependent activities of cathepsins B, L, and S as well as the pH in the lysosomal microenvironment of liver cancerous C7721 and paracancerous C7701 cells. As long as suitable labeling molecules and elemental tags are designed and synthesized, we believe that such a tandem labeling and tagging ICPMS approach can be applied to the measurement of the activities of other proteases in cells, providing more accurate information on the proteases’ biofunctions and thus implementing precise clinical diagnoses.