Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure

Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys(462) and Cys(665), as well as at Asp(339) and Glu(341) described herein for the first time. Since Cys(665) is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein.

Automated summary

The aim of this study was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. Western blot analysis confirmed the isolation of hTRPA1, and immunomagnetic separation was used to isolate hTRPA1 from transfected HEK293 cell lysates. Mass spectrometry identified modifications of hTRPA1 channel at Cys(462), Cys(665), Asp(339) and Glu(341) after SM exposure, suggesting that these modifications may play a role in hTRPA1 activation. The analytical approach described in this study can be used to predict and identify analogous adducts of hTRPA1 as targets of other SM-related chemical warfare agents.