DOI: 10.6084/m9.figshare.12330644
title: CaMKII Nav16 MS Raw Files_Ionomycin

description: Files in this folder contain raw mass spectrometry data to examine Nav1.6 phosphorylation following ionomycin treatment to promote Ca<sup>2+</sup>-dependent activation of endogenous CaMKII. A detailed description of methods can be found below.<br><br><i>Methods</i>CaMKII phosphorylation sites on Nav1.6 were examined with mass spectrometry by treating Nav1.6-expressing cells with; KN93 and tatCN21, KN92 and tatNC21Ala, ionomycin, or autophosphorylated purified aCaMKII. Autophosphorylation of CaMKII was performed in the presence of (in mM) 50 HEPES pH 7.4, 10 MgCl<sub>2</sub>, 0.5 CaCl<sub>2</sub>, 5 mM CaM, 500 mM ATP with 500 nM recombinant purified aCaMKII for 10 minutes on ice to autophosphorylate aCaMKII at Thr286. HEK293 cells stably expressing human Nav1.6 were plated onto 150 mm cell culture dishes and grown to 70% confluency prior to the following treatments. To inhibit CaMKII, cells were treated with 1 mM of the small molecule CaMKII inhibitor KN93 (Sigma-Aldrich) overnight (or the control compound KN92) at 30°C. Incubation at 30°C facilitates maximal membrane expression of the channel as previously described (6). The following day, cells were washed 3 times with PBS and incubated at 30°C with 10 mM tatCN21 (50) (or the control peptide tatCN21Ala) in HBSS for 20 minutes prior to cell lysis and immunoprecipitation (described above). To control for temperature-dependent effects, cells were also incubated with no additional treatments (naïve treatment group). To promote Ca<sup>2+</sup>-dependent activation of endogenous CaMKII, cells were treated with 10 mM ionomycin and 2 mM CaCl<sub>2</sub> for 5 minutes in HBSS prior to cell lysis and immunoprecipitation. While ionomycin treatment promotes endogenous CaMKII activity, it may also activate other Ca<sup>2+</sup>-dependent cellular kinases. Therefore, we also treated cell lysates with recombinant autophosphorylated aCaMKII <i>in vitro</i>. For this experiment, cells were similarly processed to minimize variation. The Nav1.6-antibody-bead complex was washed with an immunoprecipitation wash buffer containing (in mM) 50 HEPES, 0.1% Tween-20, 100 NaCl, 10 MgCl<sub>2</sub>, and 0.5 CaCl<sub>2</sub> to remove traces of EGTA/EDTA. Autophosphorylated aCaMKII was then added to the washed bead complex and incubated for 10 minutes at room temperature followed by 3 washes in PBS. Beads were kept in PBS prior to submission for mass spectrometry analysis. Samples were submitted to the Indiana University School of Medicine Proteomics Core Facility for sample processing (described below) and subsequent PTM analysis by nanoflow liquid chromatography coupled with electrospray ionization mass spectrometry (nanoflow LC-ESI/MS) to identify CaMKII phosphorylation sites on the channel. Following washes, the Nav1.6-antibody-bead complexes were first denatured in 8M urea and reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), followed by alkylation with 10 mM chloroacetamide. Bead complexes were then incubated with 0.5 mg of trypsin_LysC (Promega) in 2M urea overnight at 37°C. Digested peptides were injected onto an Acclaim PepMap C18 trapping column and eluted on a PepMap C18 analytical column with a linear gradient of 3% to 35% acetonitrile (in water with 0.1% formic acid) and developed over 120 minutes at room temperature at a flow rate of 700 nL/min. Effluent was electro-sprayed into Thermo Dionex UltiMate 3000 RSLC nano system and Velos Pro Orbitrap or Qexactive mass spectrometer. A blank was run prior to each injected sample to ensure there was no significant signal from solvents or the column. Raw files were analyzed using Xcaliber Qual Browser (v 2.2.48) and database searches (Proteome Discoverer v2.2, SEQUEST XCorr and Scaffold Q) against the human proteome from Uniprot (version downloaded February 15, 2017) were performed with the following parameters: a peptide mass tolerance of 10.0 ppm, fragment mass tolerance of 0.80 Da, trypsin digestion (cleavage after lysine and arginine) allowing 2 missed cleavages, carbamidomethylation of Cys was set as a fixed modification and oxidation of methionine and phosphorylation (serine, threonine, tyrosine) were considered as variable modifications. False discovery rate was set to 0.1% and peptide spectral matches were accepted if they could be established at greater than 90% probability. Results and quantitative data from each nanoflow LC-ESI/MS analysis was exported to an Excel spreadsheet (Table S1). Each MS/MS spectrum exhibiting possible phosphorylation was manually validated based on an observed 98 Da mass loss (-H<sub>3</sub>PO<sub>4</sub>) for both precursor and fragmented ions using Xcaliber Qual Browser (v.2.2.48). Phosphorylation ratios were measured by normalizing the area under the MS peak to that of the parent peptide identified in all samples for normalization across all conditions.<br>