Installing rack2 folder monitor software11/12/2022 ![]() Examples of sequential peptide degradation have unexpectedly been observed in recent oncopeptidomics studies 8 - 10. It is also difficult to measure `compound' proteolytic activities that degrade a substrate, either in unison or sequentially. Typical assays monitor substrate conversion via simple colorometric or fluorophoric read-outs 6, 7 that are easily performed in parallel but not so easily multiplexed. Proteases have also been implicated in disease, most notably in cancer where they may both promote tumor progression or suppression 3 - 5. Although they all share one principal functionality, the lysis of peptide bonds, their biological roles differ greatly, ranging from digestion of food and discarded cellular proteins to more specific functions such as basic control mechanisms in the cell, orderly progression of physiological pathways and response to trauma, to name a few 1, 2. Human cells produce well over 500 proteases 1, 2. This may be particularly true for proteolytic enzymes as they generate peptide products that are tailor-made for MS-based proteomic read-outs. As an alternative, the case could be made that, given enough substrate and time, and optimal assay conditions, catalytic or synthetic products may accumulate to levels that become readily detectable using some type of analytical technique. Furthermore, many enzymes are present below the MS detection limits in a given sample and are therefore `invisible' in traditional screens. Yet, in a typical mass spectrometry (MS)-based proteomic analysis, enzymes are simply looked at as `proteins' that may or may not be physically present in certain locations and that may undergo temporal changes in concentration. In the literal sense, activity-based or functional proteomics would therefore entail profiling based on genuine activity readouts. Enzymatic activity is bringing about chemical change in a target, ranging from simple modifications to anabolic or catabolic reactions. However, systematic measurement and quantitation of enzymatic activities in a complex biological matrix, while clearly in the realm of proteomics, is a largely unpracticed specialty at the moment. It is often stated that, from a functional or biomarker point-of-view, the analysis of proteins trumps that of mRNA (e.g., using DNA micro-arrays) since `activities' are what ultimately matter in a cell. It could be tailored to many diagnostic and pharmaco-dynamic purposes, as a read-out of catalytic and metabolic activities in body fluids or tissues. The approach may be employed for diagnostic or predictive purposes and enables profiling of 96 samples in 30 hours. ![]() Partial automation provides reproducibility and throughput essential for comparing large sample sets. Relative quantitation of the peptide metabolites is done by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. The protocol presented here describes a semi-quantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide-substrates using robotic extraction and a MALDI-TOF mass spectrometric read-out. ![]() Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. ![]()
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