An Investigation into the Properties of a Chaperone Domain and Analysis of the Non-covalently Bound Complexes with Peptide Substrates using Mass Spectrometry

2000 ◽  
Vol 28 (3) ◽  
pp. A70-A70
Author(s):  
Alison E. Ashcroft ◽  
Sheena E. Radford ◽  
Joseph E. Coyle ◽  
Maureen Pitkeathly ◽  
Ulrich F. Hartl
Keyword(s):  
Mass Spectrometry ◽  
Peptide Substrates ◽  
Covalently Bound

scholarly journals 7.87 eV laser desorption postionization mass spectrometry of adsorbed and covalently bound bisphenol a diglycidyl methacrylate

2007 ◽  
Vol 18 (6) ◽  
pp. 1097-1108 ◽  
Author(s):  
Manshui Zhou ◽  
Chunping Wu ◽  
Artem Akhmetov ◽  
Praneeth D. Edirisinghe ◽  
James L. Drummond ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bisphenol A ◽  
Laser Desorption ◽  
Covalently Bound

scholarly journals Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry

2015 ◽  
Vol 20 (5) ◽  
pp. 606-615 ◽  
Author(s):  
Juncai Meng ◽  
Ming-Tain Lai ◽  
Vandna Munshi ◽  
Jay Grobler ◽  
John McCauley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Antiviral Agents ◽  
Sampling Rate ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Label Free ◽  
Substrate Selection ◽  
Peptide Substrates ◽  
Hiv 1

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)–based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in kcat/ Km over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.

scholarly journals Versatile Method for the Detection of Covalently Bound Substrates on Solid Supports by DART Mass Spectrometry

2011 ◽  
Vol 13 (15) ◽  
pp. 3770-3773 ◽  
Author(s):  
Laura M. Sanchez ◽  
Matthew E. Curtis ◽  
Bianca E. Bracamonte ◽  
Kenji L. Kurita ◽  
Gabriel Navarro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Solid Supports ◽  
Dart Mass Spectrometry ◽  
Covalently Bound

scholarly journals A two-trick pony: lysosomal protease cathepsin B possesses surprising ligase activity

2020 ◽  
Author(s):  
Tyler R. Lambeth ◽  
Zhefu Dai ◽  
Yong Zhang ◽  
Ryan R. Julian
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Active Site ◽  
Cathepsin B ◽  
Reverse Direction ◽  
Peptide Substrates ◽  
Lysosomal Protease ◽  
Inactive Form ◽  
Step Mechanism ◽  
Protease Action

AbstractCathepsin B is an important protease within the lysosome, where it helps recycle proteins to maintain proteostasis. It is also known to degrade proteins elsewhere but has no other known functionality. However, by carefully monitoring peptide digestion with liquid chromatography and mass spectrometry, we observed synthesis of novel peptides during cathepsin B incubations. This ligation activity was explored further with a variety of peptide substrates to establish mechanistic details and was found to operate through a two-step mechanism with proteolysis and ligation occurring separately. Further explorations using varied sequences indicated increased affinity for some substrates, though all were found to ligate to some extent. Finally, experiments with a proteolytically inactive form of the enzyme yielded no ligation, indicating that the ligation reaction occurs in the same active site but in the reverse direction of proteolysis. These results clearly establish that cathepsin B can act as both a protease and ligase, although protease action eventually dominates over longer periods of time.

Analysis of Covalently Bound Polyketide Intermediates on 6-Deoxyerythronolide B Synthase by Tandem Proteolysis−Mass Spectrometry†

Biochemistry ◽  
2005 ◽  
Vol 44 (35) ◽  
pp. 11836-11842 ◽  
Author(s):  
Nathan A. Schnarr ◽  
Alice Y. Chen ◽  
David E. Cane ◽  
Chaitan Khosla
Keyword(s):  
Mass Spectrometry ◽  
Deoxyerythronolide B Synthase ◽  
Covalently Bound

scholarly journals An alternative kinase activity assay for primary cultures derived from clinical isolates

2009 ◽  
Vol 32 (2) ◽  
pp. 84
Author(s):  
Kristopher McLean ◽  
Yan Wu ◽  
Bing Siang Gan ◽  
David B O'Gorman
Keyword(s):  
Mass Spectrometry ◽  
Clinical Research ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Primary Cultures ◽  
Clinical Isolates ◽  
Mass Spectrometry Analysis ◽  
Protein Kinase Activity ◽  
Peptide Substrates ◽  
Tof Ms

Purpose: The measurement of protein kinase activity is central to understanding the signaling pathways that regulate cellular proliferation and apoptosis in virtually all disease processes. These measurements typically involve either indirect, time consuming assessment methods that require large amounts of sample, such as western immunoblotting, or the use of high maintenance, specialized equipment not typically available to a small clinical research facility. The purpose of this project was to determine if a benchtop Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) unit could be used to detect and directly assess kinase activity of the serine/threonine kinase Akt. Method: Biotinylated substrate peptides, predicted to be recognized and phosphorylated by Akt to varying extents, were incubated in crude lysates of primary cells derived directly from clinical isolates. Streptavidin-coated chips were then used to isolate the substrate peptides from the lysates after incubation. Finally SELDI-TOF-MS was used to detect the peptide substrates and identify any changes in mass resulting from phosphorylation. Results: The biotinylated peptide substrates were readily detected and a simple, rapid procedure that allows direct measurement of Akt activity in less than 1 µg of cell lysate in a 2µL volume was developed. Further, a linear correlation between native to phospho-peptide ratios and SELDI-TOF-MS output demonstrated that this approach is semi-quantitative. Conclusion: This assay avoids many of the pitfalls associated with the currently available kinase protocols as well as labour-intensive mass-spectrometry analysis by specialist laboratories. We propose that this approach may be a viable alternative for clinical research laboratories aiming to measure the activity of kinases in clinical isolates.

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