Journal
NATURE PROTOCOLS
Volume 4, Issue 8, Pages 1167-1183Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.88
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Funding
- US National Institutes of Health Grants [R33 CA111942, U24 CA126485]
- National Cancer Institute's Clinical Proteomic Technologies Initiative
- Clinical Proteomic Technology Assessment for Cancer ( CPTAC) consortium
- NATIONAL CANCER INSTITUTE [U24CA126485, R33CA111942] Funding Source: NIH RePORTER
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Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues.
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