Ltq velos pro ion trap mass spectrometer

DsbB, DsbA_C33A and DsbB-DsbA_C33A dimer (each at 100 μM) were mixed with compound 12 on ice at 1:2 ratio in 50 mM Tris buffer (pH:8) containing 300 mM NaCl and 0.05% DDM. Compound 12 was added by two increments of 100 μM. For DsbB-DsbA_C33A dimer, when absorbance at 500 nm reached a minimum upon addition of compound (around 4 minutes). Samples were directly diluted 5-fold with aqueous solution of 0.1% trifluoroacetic acid and sent for electrospray ionization mass spectrometry analysis using LTQ Velos Pro ion-trap mass spectrometer (ThermoFisher, San Jose, CA). In order to determine the site of modification, DsbB-DsbA_C33A dimer treated with compound 12 was reduced on ice with 50 mM DTT for 30 min, loaded on a HPLC column and eluted fractions corresponding to DsbB were digested with chymotrypsin. Chymotryptic peptides were subsequently pressure loaded onto a HPLC column. Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions and analyzed using HCD LTQ-Orbitrap at 15,000 resolution (ThermoFisher, San Jose, CA).