化学
多路复用
色谱法
磷脂
生物传感器
质谱法
基质金属蛋白酶
胰蛋白酶
检出限
酶
生物化学
膜
生物信息学
生物
作者
Junjie Hu,Fei Liu,Yunlong Chen,Jia Fu,Huangxian Ju
标识
DOI:10.1021/acs.analchem.3c01039
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI