Mass Spectrometry Analysis of aL58ONBY

Identity of aL58ONBY and its decaging efficiency were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC (Thermo Fisher ScientificWaltham, MA, USA) coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher ScientificWaltham, MA, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [76 (link)]. aL58ONBY was either analyzed in its “as isolated” state or after exposure to UV light (UVP BL-15; Analytik Jena US, Jena, Germany; CA 91786) for 20 min. Next, 100 pmol protein were injected and online buffer was exchanged to 200 mM ammonium acetate, pH 6.8 (AmAc) by a self-packed buffer exchange column [77 (link)] (P6 polyacrylamide gel, BioRad, Hercules, CA, USA) at a flow-rate of 100 µL per min. Mass spectra were recorded for 1000–8000 m/z at 35,000 resolution as defined at 200 m/z. The injection time was set to 200 ms. Voltages applied to the ion optics were optimized to allow for efficient ion transmission while minimizing unintentional ion activation. Only m/z corresponding to the monomer were considered for deconvolution and subsequent relative quantitation. Mass spectra were deconvoluted with UniDec version 4.0.0 beta [78 (link)] using the following processing parameters: Sample mass every 0.1 Da; peak FWHM 1 Thompson, Gaussian peak shape function.