Mass Spectrometry Analysis of Crosslinked Actin

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)].