CaMKII Nav16 MS Raw Files_Ionomycin

Files in this folder contain raw mass spectrometry data to examine Nav1.6 phosphorylation following ionomycin treatment to promote Ca²⁺-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 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>

本文件夹中的文件包含原始质谱数据，用于检测经离子霉素处理后Nav1.6的磷酸化水平，以探究内源性钙调蛋白依赖性蛋白激酶II（CaMKII, Calcium/Calmodulin-Dependent Protein Kinase II）的钙依赖性激活过程。详细的实验方法说明如下。

方法
研究通过以下方式处理表达Nav1.6的细胞，结合质谱法检测Nav1.6上的CaMKII磷酸化位点：KN93与tatCN21、KN92与tatCN21Ala、离子霉素，以及重组纯化的自磷酸化aCaMKII。
CaMKII的自磷酸化反应体系为（单位：mM）：50 HEPES（pH 7.4）、10 MgCl₂、0.5 CaCl₂、5 mM 钙调蛋白（CaM, Calmodulin）、500 mM ATP，加入500 nM重组纯化的aCaMKII，于冰上孵育10分钟，使aCaMKII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70%后进行后续处理。为抑制CaMKII，将细胞用1 mM的小分子CaMKII抑制剂KN93（Sigma-Aldrich品牌）于30℃下孵育过夜（对照组使用同浓度的KN92）。此前研究表明，30℃孵育可最大化该通道的膜表达量。次日，用PBS洗涤细胞3次，于30℃下将细胞置于含10 mM tatCN21（参考文献50）的HBSS缓冲液中孵育20分钟（对照组使用对照肽tatCN21Ala），随后进行细胞裂解与免疫沉淀（具体步骤见前文）。为控制温度依赖效应，设置未添加额外处理的空白对照组（naïve treatment group，未处理组）。
为激活内源性CaMKII的钙依赖性通路，将细胞置于含10 mM离子霉素与2 mM CaCl₂的HBSS缓冲液中孵育5分钟，随后进行细胞裂解与免疫沉淀。尽管离子霉素处理可激活内源性CaMKII，但同时也可能激活其他钙依赖性细胞激酶。因此，我们同时在体外对细胞裂解液添加重组自磷酸化aCaMKII进行对照实验。本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洗涤，该缓冲液组成如下（单位：mM）：50 HEPES、0.1% Tween-20、100 NaCl、10 MgCl₂、0.5 CaCl₂，以去除痕量的EGTA/EDTA。随后向洗涤后的磁珠复合物中加入自磷酸化的aCaMKII，于室温下孵育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。
样本提交至印第安纳大学医学院蛋白质组学核心设施进行样本处理（具体步骤见下文），随后通过纳流液相色谱-电喷雾电离质谱法（nanoflow LC-ESI/MS, nanoflow Liquid Chromatography-Electrospray Ionization Mass Spectrometry）进行后续的翻译后修饰（PTM, Post-Translational Modification）分析，以鉴定该通道上的CaMKII磷酸化位点。
洗涤完成后，先将Nav1.6抗体-磁珠复合物在8M尿素中变性，并用5 mM 三(2-羧乙基)膦盐酸盐（TCEP, Tris(2-carboxyethyl)phosphine hydrochloride）进行还原，随后用10 mM 氯乙酰胺进行烷基化。接着将磁珠复合物与0.5 mg 胰蛋白酶-LysC（Promega品牌）置于2M尿素中，于37℃下孵育过夜进行酶解。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%的乙腈水溶液（含0.1%甲酸）线性梯度洗脱，于室温下以700 nL/min的流速持续洗脱120分钟。洗脱液经电喷雾电离进入Thermo Dionex UltiMate 3000 RSLC nano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，以确保溶剂或色谱柱无明显信号干扰。
原始数据文件使用Xcaliber Qual Browser（版本2.2.48）进行分析，并通过数据库搜索（Proteome Discoverer v2.2、SEQUEST XCorr与Scaffold Q）比对UniProt的人源蛋白质组（2017年2月15日下载版本），搜索参数设置如下：肽段质量容忍度为10.0 ppm，碎片离子质量容忍度为0.80 Da，胰蛋白酶酶切（裂解位点为赖氨酸与精氨酸之后）允许2个漏切位点，半胱氨酸的氨基甲酰甲基化为固定修饰，甲硫氨酸的氧化与丝氨酸、苏氨酸、酪氨酸的磷酸化为可变修饰。假发现率（FDR, False Discovery Rate）设置为0.1%，肽段谱匹配的置信度需大于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）。每个显示潜在磷酸化修饰的MS/MS谱图均通过Xcaliber Qual Browser（v.2.2.48）进行人工验证，依据前体离子与碎片离子均出现98 Da的质量损失（-H₃PO₄）进行判定。磷酸化修饰的相对丰度通过将MS峰面积归一化至所有样本中均存在的母肽段峰面积，以实现不同实验条件间的标准化。