CaMKII Nav16 MS Raw Files_Preautophosphorylated aCaMKII

Files in this folder contain raw mass spectrometry data to examine Nav1.6 phosphorylation following treatment with recombinant autophosphorylated aCaMKII <i>in vitro</i>. A detailed description of methods can be found below.<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 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 <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 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.<br>本文件夹内的文件包含用于检测**体外（in vitro）**经重组自磷酸化α钙/钙调蛋白依赖性蛋白激酶II（αCaMKII）处理后Nav1.6磷酸化状态的原始质谱（mass spectrometry）数据。详细实验方法详见下文。 实验方法 针对Nav1.6的CaMKII磷酸化位点，本研究通过质谱分析进行检测：将表达Nav1.6的细胞分别用以下试剂组合处理：KN93与tatCN21、KN92与tatCN21Ala、离子霉素（ionomycin），或重组纯化的自磷酸化αCaMKII。 CaMKII的自磷酸化反应体系（终浓度单位为mM）如下：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，培养至细胞汇合度达70%后开展后续处理。为抑制CaMKII活性，细胞用1 mM的小分子CaMKII抑制剂KN93（Sigma-Aldrich产品）于30℃孵育过夜，对照组则使用同家族对照化合物KN92。既往研究表明，30℃孵育可最大化该离子通道的膜表达量（文献6）。 次日，用磷酸盐缓冲液（PBS）洗涤细胞3次，随后将细胞置于含10 mM tatCN21（参考文献50）的汉克斯平衡盐溶液（HBSS）中，于30℃孵育20分钟；对照组则使用对照肽tatCN21Ala。孵育结束后进行细胞裂解与免疫沉淀（方法如前所述）。为控制温度依赖的非特异性效应，同时设置未添加任何处理的空白对照组（naïve treatment group，即未处理组）。 为激活内源性CaMKII的钙依赖性活性，将细胞置于含10 mM离子霉素与2 mM CaCl₂的HBSS中孵育5分钟，随后进行细胞裂解与免疫沉淀。需注意，离子霉素处理虽可激活内源性CaMKII，但也可能同时激活其他钙依赖性细胞激酶。因此，本研究还在体外对细胞裂解液施加了重组自磷酸化αCaMKII处理。本次实验中，所有样本的处理流程保持一致，以尽可能减少实验误差。 将Nav1.6-抗体-磁珠复合物用免疫沉淀洗涤缓冲液（终浓度单位为mM：50 mM HEPES、0.1% Tween-20、100 mM NaCl、10 mM MgCl₂、0.5 mM CaCl₂）洗涤，以去除残留的EGTA/EDTA。随后向洗涤后的复合物中加入自磷酸化的αCaMKII，于室温孵育10分钟，再用PBS洗涤3次。磁珠在提交质谱分析前需保存在PBS中。 样本送至印第安纳大学医学院蛋白质组学核心实验室进行样本前处理（方法详见下文），随后采用纳流液相色谱-电喷雾电离质谱（nanoflow liquid chromatography coupled with electrospray ionization mass spectrometry, nanoflow LC-ESI/MS）进行后续的翻译后修饰（post-translational modification, PTM）分析，以鉴定该离子通道上的CaMKII磷酸化位点。 洗涤后的Nav1.6-抗体-磁珠复合物先用8 M尿素变性，再用5 mM三(2-羧乙基)膦盐酸盐（TCEP）进行还原，随后用10 mM氯乙酰胺进行烷基化。之后将磁珠复合物与0.5 mg胰蛋白酶-赖氨酸C（trypsin_LysC，Promega产品）置于2 M尿素中，于37℃孵育过夜进行酶解。酶解后的肽段被注入Acclaim PepMap C18捕集柱，随后在PepMap C18分析柱上以3%至35%乙腈（含0.1%甲酸的水溶液）的线性梯度进行洗脱，于室温下以700 nL/min的流速分离120分钟。洗脱液经电喷雾电离进入Thermo Dionex UltiMate 3000 RSLC nano纳流液相系统与Velos Pro Orbitrap或Q Exactive质谱仪。每个样本进样前均运行空白对照，以确保溶剂与色谱柱无显著信号干扰。 原始数据使用Xcaliber Qual Browser（v2.2.48）进行分析，并采用Proteome Discoverer v2.2、SEQUEST XCorr与Scaffold Q软件，针对Uniprot人源蛋白质组数据库（2017年2月15日下载版本）进行数据库检索，检索参数设置如下：肽段质量偏差为10.0 ppm，碎片离子质量偏差为0.80 Da，胰蛋白酶酶切位点为赖氨酸与精氨酸残基后，允许2个漏切位点；半胱氨酸的氨基甲酰化设置为固定修饰，甲硫氨酸的氧化与丝氨酸、苏氨酸、酪氨酸的磷酸化设置为可变修饰。假发现率（FDR）设置为0.1%，肽段谱图匹配（PSM）的置信度需大于90%方可被接受。 每次纳流LC-ESI/MS分析得到的结果与定量数据均导出至Excel表格（表S1）。每一个显示潜在磷酸化修饰的MS/MS谱图均通过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-H₃PO₄）。磷酸化修饰的相对比例通过将MS峰面积归一化至所有样本中均检出的母肽段峰面积，以实现不同实验条件间的标准化。