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The whole strategy involves a series of sequential steps, that are schematised in Figure 1 and typically involves the following steps: 1) Sample collection, where biological samples are collected from the study subjects according to the experimental design, 2) Metabolite extraction, where depending on the nature of the sample and the metabolites of interest different approaches such as solvent extraction, protein precipitation and solid-phase extraction can be used, 3) Data acquisition, various analytical techniques such as GC or LC-MS are used to detect and quantify the metabolites present in the prepared samples, 4) Data preprocessing, the generated data is pre-processed for chromatographic peak detection, alignment and normalization procedures to remove sampling differences, batch effects or injection order-dependent signal drifts, 5) Data processing with bioinformatic tools for the identification and quantification of the detected metabolites and 6) Biological interpretation which involves statistical analysis to identify differences between groups and correlate metabolites with biological processes or clinical outcomes as well as to provide insights into metabolic pathways, biomarker discovery and disease mechanisms

After TMB substrate solution is added, only those wells that contain Human KLK5, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color

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10.1158/0008-5472.CAN-23-2910 196 YunevaM.ZamboniN.OefnerP.SachidanandamR.LazebnikY