Rap1 Protein Photolabeling Using Azi-Anesthetics

Photolabeling of Rap1 protein using azi-isoflurane and azi-sevoflurane was performed as previously described [16 (link)]. Azi-isoflurane and azi-sevoflurane are photoaffinity probes, developed by incorporating a diazrinyl moiety (CHN₂, 40 Da) into isoflurane and sevoflurane, respectively [17 (link)]. Azi-isoflurane or azi-sevoflurane (final concentration, 100 μM) was equilibrated with Rap1 protein (1 mg/mL) and 500 μM isoflurane or sevoflurane in a reaction volume of 300 μL for 10 min and then exposed to 300-nm light under a Rayonet RPR-3000 Lamp (Southern New England Ultraviolet Co.; Branford, CT, USA) in a 1-mm path-length quartz cuvette for 25 min. The protein was separated on an SDS-polyacrylamide gel and stained with Coomassie G-250. The protein gel band was excised for liquid chromatography (LC)-mass spectrometry (MS)/MS. After trypsin digestion, samples were injected into a nano-LC column with online electrospray into a LTQ linear ion trap (Thermo Fisher Scientific). Raw data were acquired with XCalibur (Thermo Fischer Scientific), and Sequest software (Scripps Research Institute, La Jolla, CA, USA) was used to search for b and y ions against the sequence of Rap1 for adducts of the appropriate mass (196 Da).