CaMKII Nav16 MS Scaffold Files

Data in this folder contain Scaffold files to visualize data.
The following runs are labeled accordingly:

530181 and 181= Naive
530182 and 182= CaMKII inhibition
530183 and 183= CaMKII inhibitor controls
530184 and 184= Ionomycin
530185 and 185= pre-autophosphorylated aCaMKII

Methods
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 MgCl2, 0.5 CaCl2, 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 Ca2+-dependent
activation of endogenous CaMKII, cells were treated with 10 mM ionomycin and 2 mM CaCl2 for 5 minutes in HBSS
prior to cell lysis and immunoprecipitation. While ionomycin treatment promotes
endogenous CaMKII activity, it may also activate other Ca2+-dependent
cellular kinases. Therefore, we also treated cell lysates with recombinant
autophosphorylated aCaMKII in vitro.
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 MgCl2, and
0.5 CaCl2 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 (-H3PO4)
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.