Imperial College London
Alternate

DrHelenaCocheme

Faculty of Medicine, Institute of Clinical Sciences

Honorary Senior Lecturer
 
 
 
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Contact

 

helena.cocheme Website

 
 
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Location

 

4.15ELMS BuildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@article{van:2017:10.1016/j.freeradbiomed.2017.03.037,
author = {van, Leeuwen LAG and Hinchy, EC and Murphy, MP and Robb, EL and Cocheme, HM},
doi = {10.1016/j.freeradbiomed.2017.03.037},
journal = {Free Radical Biology & Medicine},
pages = {374--382},
title = {Click-PEGylation - a mobility shift approach to assess the redox state of cysteines in candidate proteins},
url = {http://dx.doi.org/10.1016/j.freeradbiomed.2017.03.037},
volume = {108},
year = {2017}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - The redox state of cysteine thiols is critical for protein function. Whereas cysteines play an important role in the maintenance of protein structure through the formation of internal disulfides, their nucleophilic thiol groups can become oxidatively modified in response to diverse redox challenges and thereby function in signalling and antioxidant defences. These oxidative modifications occur in response to a range of agents and stimuli, and can lead to the existence of multiple redox states for a given protein. To assess the role(s) of a protein in redox signalling and antioxidant defence, it is thus vital to be able to assess which of the multiple thiol redox states are present and to investigate how these alter under different conditions. While this can be done by a range of mass spectrometric-based methods, these are time-consuming, costly, and best suited to study abundant proteins or to perform an unbiased proteomic screen. One approach that can facilitate a targeted assessment of candidate proteins, as well as proteins that are low in abundance or proteomically challenging, is by electrophoretic mobility shift assays. Redox-modified cysteine residues are selectively tagged with a large group, such as a polyethylene glycol (PEG) polymer, and then the proteins are separated by electrophoresis followed by immunoblotting, which allows the inference of redox changes based on band shifts. However, the applicability of this method has been impaired by the difficulty of cleanly modifying protein thiols by large PEG reagents. To establish a more robust method for redox-selective PEGylation, we have utilised a Click chemistry approach, where free thiol groups are first labelled with a reagent modified to contain an alkyne moiety, which is subsequently Click-reacted with a PEG molecule containing a complementary azide function. This strategy can be adapted to study reversibly reduced or oxidised cysteines. Separation of the thiol labelling step from the PEG conjugation
AU  - van,Leeuwen LAG
AU  - Hinchy,EC
AU  - Murphy,MP
AU  - Robb,EL
AU  - Cocheme,HM
DO  - 10.1016/j.freeradbiomed.2017.03.037
EP  - 382
PY  - 2017///
SN  - 1873-4596
SP  - 374
TI  - Click-PEGylation - a mobility shift approach to assess the redox state of cysteines in candidate proteins
T2  - Free Radical Biology & Medicine
UR  - http://dx.doi.org/10.1016/j.freeradbiomed.2017.03.037
UR  - http://hdl.handle.net/10044/1/46144
VL  - 108
ER  -
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