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Signal-On Mass Spectrometric Biosensing of Multiplex Matrix Metalloproteinases with a Phospholipid-Structured Mass-Encoded Microplate
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-05-26 , DOI: 10.1021/acs.analchem.3c01039 Junjie Hu 1, 2 , Fei Liu 1 , Yunlong Chen 1 , Jia Fu 2 , Huangxian Ju 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2023-05-26 , DOI: 10.1021/acs.analchem.3c01039 Junjie Hu 1, 2 , Fei Liu 1 , Yunlong Chen 1 , Jia Fu 2 , Huangxian Ju 1
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The detection of matrix metalloproteinases (MMPs) is of great importance for diagnosis and staging of cancer. This work proposed a signal-on mass spectrometric biosensing strategy with a phospholipid-structured mass-encoded microplate for assessment of multiplex MMP activities. The designed substrate and internal standard peptides were subsequently labeled with the reagents of isobaric tags for relative and absolute quantification (iTRAQ), and DSPE-PEG(2000)maleimide was embedded on the surface of a 96-well glass bottom plate to fabricate the phospholipid-structured mass-encoded microplate, which offered a simulated environment of the extracellular space for enzyme reactions between MMPs and the substrates. The strategy achieved multiplex MMP activity assays by dropping the sample in the well for enzyme cleavages, followed by adding trypsin to release the coding regions for ultrahigh performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) analysis. The peak area ratios of released coding regions and their respective internal standard (IS) peptides exhibited satisfied linear ranges of 0.05–50, 0.1–250, and 0.1–100 ng mL–1 with the detection limits of 0.017, 0.046, and 0.032 ng mL–1 for MMP-2, MMP-7, and MMP-3, respectively. The proposed strategy demonstrated good practicability in inhibition analysis and detections of multiplex MMP activities in serum samples. It is of great potential for clinical applications and can be expanded for multiplex enzyme assays.
中文翻译：
使用磷脂结构质量编码微孔板对多重基质金属蛋白酶进行信号质谱生物传感
基质金属蛋白酶（MMP）的检测对于癌症的诊断和分期非常重要。这项工作提出了一种使用磷脂结构质量编码微板的信号质谱生物传感策略，用于评估多重 MMP 活性。设计好的底物和内标肽随后用同量异位标签试剂进行相对和绝对定量（iTRAQ）标记，并将DSPE-PEG（2000）马来酰亚胺包埋在96孔玻璃底板表面以制备磷脂-结构化质量编码微孔板，为 MMP 和底物之间的酶反应提供模拟的细胞外空间环境。该策略通过将样品滴入孔中进行酶裂解来实现多重 MMP 活性测定，然后添加胰蛋白酶释放编码区，用于超高效液相色谱-串联质谱 (UHPLC-MS/MS) 分析。释放的编码区及其各自的内标 (IS) 肽的峰面积比表现出令人满意的线性范围：0.05–50、0.1–250 和 0.1–100 ng mL–1， MMP-2、MMP-7 和 MMP-3的检测限分别为 0.017、0.046 和 0.032 ng mL –1 。该策略在血清样品中多重MMP活性的抑制分析和检测中表现出良好的实用性。它具有巨大的临床应用潜力，并且可以扩展到多重酶测定。
更新日期：2023-05-26
中文翻译：
使用磷脂结构质量编码微孔板对多重基质金属蛋白酶进行信号质谱生物传感
基质金属蛋白酶（MMP）的检测对于癌症的诊断和分期非常重要。这项工作提出了一种使用磷脂结构质量编码微板的信号质谱生物传感策略，用于评估多重 MMP 活性。设计好的底物和内标肽随后用同量异位标签试剂进行相对和绝对定量（iTRAQ）标记，并将DSPE-PEG（2000）马来酰亚胺包埋在96孔玻璃底板表面以制备磷脂-结构化质量编码微孔板，为 MMP 和底物之间的酶反应提供模拟的细胞外空间环境。该策略通过将样品滴入孔中进行酶裂解来实现多重 MMP 活性测定，然后添加胰蛋白酶释放编码区，用于超高效液相色谱-串联质谱 (UHPLC-MS/MS) 分析。释放的编码区及其各自的内标 (IS) 肽的峰面积比表现出令人满意的线性范围：0.05–50、0.1–250 和 0.1–100 ng mL–1， MMP-2、MMP-7 和 MMP-3的检测限分别为 0.017、0.046 和 0.032 ng mL –1 。该策略在血清样品中多重MMP活性的抑制分析和检测中表现出良好的实用性。它具有巨大的临床应用潜力，并且可以扩展到多重酶测定。
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