Development of inductively coupled plasma-mass spectrometry-based protease assays.
Development of inductively coupled plasma-mass spectrometry-based protease assays.
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DOI：
10.1016/j.ab.2009.11.010
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发表时间：
2010-03-01
影响因子：
2.9
通讯作者：
Nitz M
中科院分区：
文献类型：
--
作者：
Lathia US;Ornatsky O;Baranov V;Nitz M
快速、灵敏、定量检测蛋白酶对药物开发和疾病诊断具有重要意义。本文描述了一种使用电感耦合等离子体质谱（ICP-MS）检测的蛋白酶活性测定方法。肽α-胰凝乳酶底物在n端含有镧系离子螯合物以提供独特的元素标记。生物素标签被附加到肽的c端，允许从酶消化中分离未切割的肽。通过ICP-MS定量测定肽裂解产物的镧系离子信号来测定酶活性。合成的生物素化底物包括Lu-DTPA- asp - leu - leu - val - tyr ~ Asp-Lys（生物素）和Lu-DTPA-βAla-βAla-βAla-βAla -βAla-βAla-βAla- gly - ser - ala - tyr ~ Gly-Lys-Arg-Lys（生物素）-酰胺。用市售的荧光底物（如- aapf - amc）平行测定α-凝乳胰蛋白酶进行比较。使用ICP-MS法可以很容易地检测到低至2pm的酶，这优于使用α-凝乳胰蛋白酶荧光底物（如- aapf - amc）的检测限。此外，我们证明了使用这种方法来检测HeLa细胞裂解物中的凝乳胰蛋白酶活性。
Rapid, sensitive and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here, an assay for protease activity which uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N-terminus to provide a distinct elemental tag. A biotin label was appended to the C-terminus of the peptide allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(Biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay enzyme concentrations as low as 2 pM could be readily detected which was superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.
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影响因子：
2.9
作者：
BAUER, CA;THOMPSON, RC;BLOUT, ER
通讯作者：
BLOUT, ER
影响因子：
4.9
作者：
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影响因子：
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作者：
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