CaMKII Nav16 MS Raw Files_CaMKII Inhibition

Files in this folder contain raw mass spectrometry data to examine Nav1.6 phosphorylation following CaMKII inhibition with the inhibitors KN93 and tatCN21. A detailed description of methods can be found below. 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.本文件夹内的文件为原始质谱(mass spectrometry)数据，用于探究使用抑制剂KN93与tatCN21抑制钙调蛋白依赖性蛋白激酶II（CaMKII）后Nav1.6的磷酸化水平。详细的实验方法说明如下。 Methods CaMKII磷酸化位点的检测采用质谱技术完成，具体方式为对表达Nav1.6的细胞施加以下处理：抑制剂KN93与tatCN21、阴性对照化合物KN92与tatNC21Ala、离子霉素（ionomycin），以及自磷酸化纯化的αCaMKII。 CaMKII的自磷酸化反应体系如下（终浓度单位：mM）：50 mM HEPES（pH 7.4）、10 mM MgCl2、0.5 mM CaCl2、5 mM 钙调蛋白（CaM）、500 mM ATP，加入500 nM重组纯化的αCaMKII，置于冰上反应10分钟，使αCaMKII在Thr286位点发生自磷酸化。 将稳定表达人源Nav1.6的HEK293细胞接种于150 mm细胞培养皿中，培养至汇合度达70%后进行后续处理。为抑制CaMKII，将细胞置于30℃下，用1 mM的小分子CaMKII抑制剂KN93（Sigma-Aldrich品牌）孵育过夜，同时以阴性对照化合物KN92作为平行对照。如先前文献所述(6)，30℃孵育可使该离子通道的膜表达量达到峰值。 次日，用磷酸盐缓冲液（PBS）洗涤细胞3次，随后将细胞置于HBSS缓冲液中，于30℃下加入10 mM tatCN21（参考文献50）孵育20分钟，阴性对照为同浓度的对照肽tatCN21Ala，之后进行细胞裂解与免疫沉淀（方法见前文）。为控制温度依赖的实验效应，设置无额外处理的空白对照组（naïve treatment group）。 为激活内源性CaMKII的Ca²+依赖通路，将细胞置于HBSS缓冲液中，用10 mM离子霉素与2 mM CaCl2孵育5分钟，随后进行细胞裂解与免疫沉淀。尽管离子霉素可提升内源性CaMKII的活性，但同时也可能激活其他Ca²+依赖的细胞激酶。为此，我们还在体外体系中向细胞裂解液中加入重组自磷酸化的αCaMKII进行处理。 为尽可能减少实验误差，本实验各组细胞采用统一的处理流程。将Nav1.6抗体-磁珠复合物用免疫沉淀洗涤缓冲液进行洗涤，该缓冲液成分如下（终浓度单位：mM）：50 mM HEPES、0.1% Tween-20、100 mM NaCl、10 mM MgCl2、0.5 mM CaCl2，以去除残留的EGTA与EDTA。 随后将自磷酸化的αCaMKII加入洗涤后的抗体-磁珠复合物中，于室温下孵育10分钟，再用PBS洗涤3次。将磁珠保存在PBS中，待送至质谱平台进行分析。 Samples 样本被送至印第安纳大学医学院蛋白质组学核心设施（Indiana University School of Medicine Proteomics Core Facility）进行样本处理（说明如下），随后采用纳流液相色谱-电喷雾电离质谱（nanoflow LC-ESI/MS）技术进行翻译后修饰（PTM，Post-translational Modification）分析，以鉴定Nav1.6通道上的CaMKII磷酸化位点。 洗涤完成后，先将Nav1.6抗体-磁珠复合物置于8 M尿素中进行变性，再用5 mM 三(2-羧乙基)膦盐酸盐（TCEP）进行还原反应，随后用10 mM 氯乙酰胺进行烷基化修饰。将磁珠复合物置于2 M尿素体系中，加入0.5 mg 胰蛋白酶-赖氨酸C（trypsin_LysC，Promega品牌），于37℃下孵育过夜。 将酶解得到的肽段注入Acclaim PepMap C18捕集柱，随后在PepMap C18分析柱上进行分离洗脱：流动相为含0.1%甲酸的水溶液与乙腈的混合液，采用3%至35%的乙腈线性梯度洗脱，洗脱时长为120分钟，室温下流速控制为700 nL/min。洗脱液经电喷雾电离后进入Thermo Dionex UltiMate 3000 RSLC nano液相系统与Velos Pro Orbitrap或Qexactive质谱仪进行检测。 每个样本进样前均运行空白对照，以确保溶剂与色谱柱无明显信号干扰。原始质谱数据采用Xcaliber Qual Browser（v2.2.48）进行分析，并基于Uniprot数据库下载的2017年2月15日版人源蛋白质组，使用Proteome Discoverer v2.2、SEQUEST XCorr与Scaffold Q软件进行数据库检索。检索参数设置如下：肽段质量误差容忍度为10.0 ppm，碎片离子质量误差容忍度为0.80 Da；胰蛋白酶酶切规则为在赖氨酸与精氨酸残基后切割，允许最多2个漏切位点；半胱氨酸的氨基甲酰化设为固定修饰，甲硫氨酸的氧化以及丝氨酸、苏氨酸、酪氨酸的磷酸化设为可变修饰。 错误发现率（FDR，False Discovery Rate）设置为0.1%，当肽段谱匹配的置信度大于90%时，判定为有效匹配。将每次纳流LC-ESI/MS分析得到的结果与定量数据导出至Excel表格（补充表S1）。 针对每一条显示存在磷酸化修饰的MS/MS谱图，采用Xcaliber Qual Browser（v2.2.48）进行手动验证：验证依据为前体离子与碎片离子均出现98 Da的质量损失（即脱去H3PO4）。磷酸化水平的比值通过将质谱峰面积归一化至所有样本中均存在的母肽段峰面积来计算，以实现不同实验条件间的定量归一化。