Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry Journal Article
| Authors: | Villanueva, J.; Nazarian, A.; Lawlor, K.; Tempst, P. |
| Article Title: | Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry |
| Abstract: | 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. |
| Keywords: | reproducibility; proteome; diagnostic procedure; metastasis; pharmacodynamics; in vitro study; enzyme activity; proteomics; quantitative analysis; thyroid cancer; robotics; matrix assisted laser desorption ionization time of flight mass spectrometry; software; enzyme assay; peptide hydrolases; peptide synthesis; body fluid; enzyme active site; peptidase; functional proteomics; metabolic activation; spectrometry, mass, matrix-assisted laser desorption-ionization |
| Journal Title: | Nature Protocols |
| Volume: | 4 |
| Issue: | 8 |
| ISSN: | 1754-2189 |
| Publisher: | Nature Publishing Group |
| Date Published: | 2009-01-01 |
| Start Page: | 1167 |
| End Page: | 1183 |
| Language: | English |
| DOI: | 10.1038/nprot.2009.88 |
| PUBMED: | 19617888 |
| PROVIDER: | scopus |
| PMCID: | PMC2760592 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 3" - "Export Date: 30 November 2010" - "Source: Scopus" |
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324Tempst -
9Lawlor -
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