Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.
Anal Chem. 2009 Dec 15;81(24):9961-71. doi: 10.1021/ac901703g.
Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.
基质金属蛋白酶（MMPs）是一类内肽酶，可分解细胞外基质，其上调与多种疾病有关。建立了一种液相色谱/串联质谱（LC/MS/MS）方法，用于定量分析植入式微透析采样探针样品中 MMP-1 和 MMP-9 底物及其 N 端肽产物。通过对纯化的人 MMP-1 和 MMP-9 进行体外研究，优化了测定方法，并确定了广谱 MMP 抑制剂 GM 6001 的局部递送效果。局部递送 10 μM GM 6001 足以完全抑制体外产物的形成。在雄性 Sprague-Dawley 大鼠中进行了微透析探针植入皮下组织的体内研究。在微透析探针植入后立即输注 MMP-1 和 MMP-9 底物（各 50 μM），可得到约 2 μM 的恢复产物浓度。在与底物共输注 50 μM GM 6001 时，MMP-1 和 MMP-9 底物的产物形成分别减少了 30%和 25%。空白透析液对可切割 MMP 底物的酶活性呈阴性。该方法允许在体内采样过程中观察到微透析探针周围不同 MMP 的活性。
