P6 polyacrylamide gel

Sample purity, integrity, and oligomeric state was analyzed by on-line buffer exchange MS in 200 mM ammonium acetate using a Vanquish ultra-high performance liquid chromatography system coupled to a Q Exactive ultra-high mass range Orbitrap mass spectrometer (Thermo Fisher Scientific). A self-packed buffer exchange column was used (P6 polyacrylamide gel, BioRad) (44 (link)). The recorded mass spectra were deconvolved with UniDec version 4.2+ (45 (link)).

Sample purity and integrity were analyzed by online buffer exchange MS using an UltiMate™ 3000 RSLC coupled to an Exactive Plus EMR Orbitrap instrument (Thermo Fisher Scientific, Grand Island, NY, USA) modified to incorporate a quadrupole mass filter and allow for surface-induced dissociation [81 (link)]. Between 100 and 300 pmole protein (referring to monomer) were injected and online buffer exchanged to 200 mM ammonium acetate, pH 6.8 by a self-packed buffer exchange column [81 (link),82 (link)] (P6 polyacrylamide gel; Bio-Rad Laboratories, Hercules, CA, USA) at a flow rate of 100 µL per min. Mass spectra were recorded for 1000–14000 m/z at 8750 resolution, as defined at 200 m/z. The injection time was set to 200 ms. Voltages applied to the transfer optics were optimized to allow ion transmission while minimizing unintentional ion activation. Mass spectra were deconvoluted with UniDec version 4.0.0 beta, England [83 (link)].

G-actin (20 μM) was cross-linked by addition of ACD_Vc (10 nM) followed by pelleting either in the absence or presence of CFL2 or PHD as described above. Supernatant fractions were collected and analyzed using mass spectrometry. 1-μL samples were injected onto a self-packed buffer exchange column (P6 polyacrylamide gel, Bio-Rad Laboratories, Hercules, CA, USA) and buffer exchanged into 200 mM ammonium acetate (pH 6.8) at a flow rate of 100 μL/min using a Vanquish UHPLC (Thermo Fisher Scientific, Waltham, MA, USA) [56 (link)]. Samples were ionized using a heated electrospray ionization (HESI) source with a spray voltage of 3.75 kV and source temperature of 275 °C. Samples were sprayed into a Q Exactive ultra-high mass range (UHMR) instrument modified with a surface induced dissociation (SID) device (not used in this study) [57 (link)]. Mass spectra were collected at 1000 to 16,000 m/z range at 6000 resolution (at 400 m/z). In-source trapping of 200 V and higher-energy collision dissociation (HCD) of 120 V were applied to remove adducts; harsh conditions were considered acceptable because of the covalent nature of the analyte. Acquired spectra were averaged across the elution time of the protein. Deconvolution of the averaged spectrum and mass determination was accomplished manually and/or using UniDec software [58 (link)].

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.