人外周血中的MMP-2和MMP-9：优化明胶酶校正剂用于降解研究和发现新型明胶分解酶。,Journal of Proteome Research

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人外周血中的MMP-2和MMP-9：优化明胶酶校正剂用于降解研究和发现新型明胶分解酶。
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2019-10-30 , DOI: 10.1021/acs.jproteome.9b00261
Daniela Ligi ₁ , Rosanna Maniscalco ₁ , Ferdinando Mannello ₁

Affiliation

基质金属蛋白酶2和-9（分别为明胶酶A和B）是与多种生理病理状况密切相关的酶。明胶酶谱法被认为是同时测定明胶酶的酶原，活性和复合形式的主要定性/半定量测定法之一。关键步骤由样品收集方法，分子量标准校准物和不同酶谱测定规程的变化代表。需要对这些方面进行标准化，以减少技术程序之间的差异并解释不同实验室之间的结果。在这项研究中，我们描述了一种具有增加的孔径的明胶酶谱学的新方案，它改善了不同分子量的明胶酶的分离。还报道了一种通过从外周血中提取MMP-2和MMP-9来获得用于明胶酶谱分析的明胶酶校正剂的新方法。我们的方法提供了一种明胶酶校准物，该校准物在室温下和多次冻融循环中均具有增强的稳定性。该校准物制剂也适用于体外翻译后修饰。改进的酶谱方法首次使我们能够在人外周血样本中揭示以非常高的分子量解析的新的明胶分解带，可能是MMP-9的复合物，这是经典酶谱方法无法检测到的。该校准物制剂也适用于体外翻译后修饰。改进的酶谱方法首次使我们能够在人外周血样品中揭示以非常高的分子量解析的新的明胶分解带，可能是MMP-9的复合物，而经典酶谱方法无法检测到。该校准物制剂也适用于体外翻译后修饰。改进的酶谱方法首次使我们能够在人外周血样本中揭示以非常高的分子量解析的新的明胶分解带，可能是MMP-9的复合物，这是经典酶谱方法无法检测到的。

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MMP-2 and MMP-9 in Human Peripheral Blood: Optimizing Gelatinase Calibrator for Degradome Research and Discovering a Novel Gelatinolytic Enzyme.

Matrix metalloprotease-2 and -9 (gelatinase A and B, respectively) are enzymes crucially involved in a plethora of physiopathological conditions. Gelatin zymography is considered one of the major qualitative/semiquantitative assays for simultaneously determining zymogenic, active, and complexed forms of gelatinases. Critical steps are represented by variations in sample collection methods, molecular weight standard calibrators, and different zymography assay protocols. A normalization of these aspects is required for reducing discrepancies in technical procedures and interpreting results among different laboratories. In this study, we describe a novel protocol for gelatin zymography with increased pore size, which improves the separation of gelatinases with different molecular weights. A new method for obtaining gelatinase calibrator for gelatin zymography, by extracting MMP-2 and MMP-9 from peripheral blood, is also reported. Our method provides a gelatinase calibrator with enhanced stability both at room temperature and during multiple freeze-thaw cycles. This calibrator preparation is also suitable for in vitro post-translational modifications. For the first time, the improved zymography protocol allowed us to reveal in human peripheral blood samples new gelatinolytic bands resolved at very high molecular weight, likely complexes of MMP-9, undetectable with classical zymography protocols.

更新日期：2019-10-30

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