FIG. 5.

MS of FNR from aerobically grown bacteria after alkylation by NEM in vivo. E. coli CAG627(pMW68) with induced GST-FNR production (OD₅₇₈ of 1.0) was grown under inducing conditions and labeled by adding NEM (10 mM) to the medium for 15 min without permeabilization of the bacteria. FNR was isolated and separated from GST by thrombin. FNR (600 μg/ml) was subjected to reversed-phase HPLC on a C₁₈ column connected online to the MS instrument. Unmodified FNR has a mass of 28,111 Da (cleavage product of FNR-GST); modified forms with one, two, three, four, and five NEM residues have predicted masses of 28,235, 28,359, 28,483, 28,607, and 28,781 Da, respectively.