Inhibitory Covalent Labeling and Clickable-Eu-Tagging-Based ICPMS: Measurement of pH-Dependent Absolute Activities of the Cathepsins in Hepatocyte Lysosomes
- Caixia JiCaixia JiDepartment of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, ChinaMore by Caixia Ji
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- Yong LiangYong LiangDepartment of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, ChinaMore by Yong Liang
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- Fuchun GeFuchun GeDepartment of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, ChinaMore by Fuchun Ge
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- Limin Yang*Limin Yang*E-mail: [email protected] (L.M.Y.).Department of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, ChinaMore by Limin Yang
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- Qiuquan Wang*Qiuquan Wang*E-mail: [email protected] (Q.Q.W.).Department of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, ChinaState Key Lab of Marine Environmental Science, Xiamen University, Xiamen 361005, ChinaMore by Qiuquan Wang
Abstract
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 using 153Eu-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.
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