Identification of active center (modified)cysteine in three Zobellia galactanivorans sulfatases

LC-MS/MS analysis 50 µg of purified protein were diluted to a total volume of 100 µL with 50 mM triethylammonium bicarbonate (TEAB) buffer. Samples were first digested with trypsin (500 ng) for 5 h at 37°C and 1,100 rpm, which was followed by AspN digestion (500 ng) at 27°C and 1,100 rpm. In the case of AspN, ZnCl2 was added at a final concentration of 2.5 mM using a 100 mM stock solution in 1 M TEAB. The reaction was stopped by adding 10 µl HCl. Peptides were extracted using C18 tips (C18 tips, Pierce, Thermo Fisher Scientific) using solvent A (0.1 % acetic acid in water) and solvent B (0.1 % acetic acid in acetonitrile). 200 µL of solvent A were added to samples. C18 material was equilibrated (3 % solvent B in solvent A) before samples were loaded. After washing with solvent A, peptides were eluted (200 µL 60 % solvent B in solvent A). Samples were dried completely using a SpeedVac and resuspended in 20 µL solvent A containing retention time calibration peptides (1x iRT, Biognosys). Samples were analysed by parallel reaction monitoring (PRM) on a Q Exactive instrument coupled with an EASY-nLC 1000 (both Thermo Fisher Scientific). Peptides were loaded onto an in-house built C18 column in solvent A and eluted with a 45 min gradient of 1-38 % solvent B at a flow rate of 300 nL min-1 for subsequent MS/MS analysis. MS1 survey scans were recorded in the Orbitrap (70,000 resolution at m/z = 200, 3e6 AGC target, max IT 200 ms, mass range 200 to 1800 m/z). For PRM, we used isolation lists of ~40 ions per run, which included peptides covering the active center cysteine, peptide lengths <35, charge states of 2-4 and respective modifications at the cysteine of interest (serine -15.977156, formylglycine aldehyde -17.992805, formylglycine diol +0.017759). Transitions were analyzed with Skyline v23.1.0.268 (https://doi.org/10.1002/mas.21540). High-selectivity extraction was enabled. Transitions were evaluated manually: For a spectrum to be accepted, the modification site needed to be covered by the smallest possible fragment ion as well as by three fragments of an ion series larger than the modification site (y-ions mandatory and b-ions optional). In addition, transitions with a mass error ≥ 5 ppm were discarded.

Three carrageenan-active sulfatases from Zobellia galactanivorans: ZG_3145 = ZgCgsA ZG_3146 = ZgCgsB1 ZG_3151 = ZgCgsC