Investigating redox posttranslational modifications in proteins using mass spectrometry
Abstract
Redox potential, a measure of how oxidising or reducing an environment is, is tightly
regulated by cells to minimise detrimental chemical oxidation and reduction reactions.
In proteins, it is the sulfur containing cysteine residues that can be post-translationally
modified through specific redox reactions, for example, the formation of disulfide
bonds between cysteine residues can be crucial to protein structure. It has recently
been hypothesised that signalling pathways utilising redox regulated proteins may be
arranged into electrochemical series.
The characterisation of the redox properties of specific cysteine residues in proteins
has proven difficult using traditional redox characterisation methods such as cyclic
voltammetry. A number of biochemical methods have been developed for studying the
effect of the redox environment on proteins, many making use of mass spectrometry
and allowing for localisation of the site of the modification to specific cysteine residues.
However, fewer methods have been reported that facilitate accurate quantification for
the determination of the mid-point potential of these redox regulated cysteine residues.
Here, a differential labelling protocol using high resolution mass spectrometry techniques
for the study of redox chemistry of cysteine residues in proteins will be reported.
The protocol exploits the novel chemistry of thiol groups for specific alkylation and
allows for both qualitative and quantitative experiments. Thioredoxin-1 from E. coli
and human systems was used as a model protein and a novel disulfide bond was
characterised. The reducing potential of the active site cysteine residues of human
thioredoxin were found to be very similar to those of the E. coli proteoform, -276 ±
1 and -281.4 ± 0.3 mV respectively. The remaining three cysteine residues of human
thioredoxin were found to be regulated at more oxidising potentials.
The protocol developed was applied to a protein from the cell death pathway of apoptosis;
human caspase-3 is an executioner protease from the caspase cascade. Caspase-3
was found to contain three redox sensitive cysteine residues. The catalytically active
cysteine residue was redox regulated via two mechanisms, glutathionylation and disulfide
bond formation. One of these mechanisms gives the active site cysteine residue
a calculated reducing potential of -165 ± 6 mV supporting the correlation between
caspase-3 activity and its observed role in the apoptotic pathway but not in necrotic
cell death.