Agilent 6420 triple quadrupole

Mass scanning and fragmentation were acquired by direct injection in Agilent 6420 triple quadrupole (Agilent, LC-MS QQQ 6420). The MS was run in the positive ion mode with 10 kV as capillary voltage. Source temperature was adjusted at 200°C. Meanwhile, highly pure nitrogen was utilized as auxiliary and sheath gas at flow rates of 40 and 80 arb. unit, respectively. MS/MS fragmentation was conducted at collision energy of 15 ev. In a full scan mode, ions were traced within 50–2000 m/z mass range. NMR was conducted on Bruker High Performance Avance III (400 MHz) NMR spectrometer. One dimensional ¹H- and ¹³C-NMR as well as two dimensional HMBC and HSQC were performed. NMR data are recorded in S1 Table while HMBC and NOESY correlations are illustrated in S2 Fig. ESIMS (positive ion mode) m/z (rel. int.): 247.14 [M+H]⁺, (100); 229.23 [M+H-18 (H₂O)]⁺, (23); 201.05 [M+H-(H₂O + CO)]⁺, (28); 173.05 [M+H-(H₂O + 2CO)]⁺, (38); 159 (11); 92.82 (14). Data comply with the previously published literature [9 , 15 (link)].

The sulfoacetaldehyde product of isethionate oxidation with BkTauF was detected by derivatization with 2,4-dinitrophenylhydrazine (DNPH) (J&K). A 200 μl reaction mixture, containing 50 mM CAPSO, pH 10.0, 5 μg BkTauF, 0.1 M isethionate and 1 mM NAD⁺, was incubated for 10 min at 30°C. Two negative controls, omitting either BkTauF or isethionate, were also prepared. One hundred microliters of reaction solution was mixed with 1.1 ml of sodium acetate solution (0.73 M), then 800 μl DNPH (0.04%) solution was added. The mixture was incubated at 50°C for 1 h and then filtered prior to LC-MS analysis.
LC-MS analysis was performed on an Agilent 6420 Triple Quadrupole LC/MS instrument (Agilent Technologies), on an Agilent ZORBAX SB-C18 column (4.6 × 250 mm). The column was equilibrated with 75% of 0.1% formic acid in H₂O, 25% of 0.1% formic acid in CH₃CN, and developed at a flow rate of 1.0 ml/min from 25 to 65% CH₃CN. UV detection was set at 360 nm.

Detection of IseH catalyzed SAM cleavage and product formation using LC-MS assays were performed as described elsewhere^{47 (link)}. A reaction mixture (500 μL total volume) containing 20 mM Tris/HCl, pH 7.5, 100 mM KCl, 200 μM Ti(III) citrate, and 20 μM reconstituted MBP-IseH was incubated for 15 min at RT in the glovebox to allow the reduction of IseH. SAM (Sigma, 0.5 mM final concentration) was added to initiate the cleavage reaction. A control assay omitting Ti (III) was also performed. The reaction was incubated at RT in the glovebox overnight and quenched with formic acid (5% v/v final concentration). The reaction mixture was then incubated in a boiling water-bath for 45 s to completely denature the protein. The precipitated protein was removed by centrifugation at 14,000 × g for 10 min. and the supernatant was filtered through a 0.22-μm PES membrane. A 20 μL portion of the supernatant was analyzed by an Agilent 6420 Triple Quadrupole LC/MS instrument (Agilent Technologies) on a C18 column (Advantage ECHELON C18 4 μm 150 × 2.1 mm P/N: ADV8010, manufactured by ANALYTICAL). The solvent system consisted of water (A) and acetonitrile (B), and the sample was eluted with a linear gradient of 0–16% B over 30 min, with a flow rate of 0.5 mL/min. The products were detected by UV absorption at 257 nm, and 5′-dA was compared to commercial standard and verified by mass spectrometry.