这项工作得到了 nih 国家糖尿病和消化系统和肾脏疾病研究所-NIDDK 赠款 R24 DK085610 (PI: e 沃丁) 的支持。JGM 得到了 NIH T32 奖学金 (T32 AG000266, PI: j 坎皮西) 的支持。提交人承认, 在降压研究所 (1S10 OD016281, PI: B.W. 吉布森) 的 TripleTOF 6600 系统中, NIH 共享的仪器赠款支持。

1. 用同位素标记的醋酸或琥珀酸酸酐定量 Acetylate 和/或琥珀酸蛋白
<ol><li>用尿素和铵碳酸盐 (TEAB) 溶液 (8 米尿素, 200 毫米 TEAB, pH 8), 在平行时, 3 复制100µg 的牛血清白蛋白 (BSA) 蛋白样品, 浓度为1µg/µL (总100µL)。 注意: 使用无胺缓冲是很重要的。在代表性结果部分使用的蛋白质样品是 bsa, 定量地琥珀酰化 bsa 和它的混合物产生 bsa 样品与定义的琥珀酰化居住在 0%, 1%, 10%, 50% 和100% 分别 (每个样品将准备在3复制)。该协议还使用了来自大肠杆菌和小鼠肝脏的蛋白质裂解物 4. 该协议也可以应用于其他细胞或组织裂解物。</li>
	<li>减少100µL 的 BSA 样品 (100 µg) 与8µL 250 毫米 dithiothreitol (德勤) 达到最终浓度的20毫米, 并孵化样品在37°c 30 摄氏度与鼓动在 1400 rpm。</li>
	<li>烷基化减少的 BSA 样品与21.6 µL 200 毫米 iodoacetamide 达到最后的集中40毫米 iodoacetamide 和孵化样品在黑暗中在室温下为30分钟。 注意: 重要的是要有 iodoacetamide 浓度略高于2x 的多点, 以确保所有还原蛋白硫醇烷基化。</li>
	<li>
继续准备醋酸酸酐-d6 (步骤 1.4.1) 或琥珀酸酸酐-d4 (步骤 1.4.2), 用于化学 (定量) 乙酰化或琥珀酰化的样品。
	<ol>
<li>从分子量 (108.13 克/摩尔) 和密度 (1.143 克/毫升25摄氏度) 确定1克整除的醋酸酸酐--d6 溶液的浓度 (M)。 注: 1 克包包含9248µmol 醋酸酐-d6 在875µL 体积, 产生一个10.57 米溶液用于每乙酰化的样品。</li>
		<li>在反应前立即将无水二甲基亚砜中的粉末溶解, 以避免试剂水解, 制备 5 M 溶液的琥珀酸酸酐-d4 。在461µL 无水二甲基亚砜中溶解0.24 克琥珀酸酸酐-d4 , 以获得5米溶液。</li>
	</ol>
</li>
	<li>分别添加60µmol 醋酸酸酐-d6 (6 µL 的 10.57 M 溶液) 或琥珀酸酸酐-d4 (12 M 解决方案) 的数量化学乙酰化或琥珀酰化, 并孵化在涡流搅拌机上取样4°c 20 分钟。 注: 由于酸酐的酸度, 蛋白质可能沉淀。注意和调整, 如步骤1.6 所述。</li>
	<li>在孵化后加入10µL 7.25 米氢氧化钠, 将 pH 值提高到 8, 以消除任何 O 酰化的副产品。通过在 ph 值纸上发现1µL, 对样品 ph 值进行简要检查。 注: 可能有必要添加超过10µL 7.25 米氢氧化钠, 以达到 pH 8。此外, 可能沉淀的蛋白质应该重新溶解。</li>
	<li>重复每酰化和 pH 调整步骤1.5 和 1.6, 总共三次。检查 pH 值并注意每重复添加的基本解决方案的体积。在上述化学酰化步骤中迅速工作, 因为酸酐会水解并失去反应性。</li>
	<li>在最终的标签反应和 pH 调整后, 添加10µL 50% 羟胺溶液, 以还原 O 酰化反应。</li>
</ol>2. 用谷氨酸 C Endoproteinase 对蛋白质样品进行消化反应
<ol><li>将尿素浓度稀释至0.8 米, 使用50毫米 TEAB, pH 8, 并使样品体积正常化。</li>
	<li>通过添加 endoproteinase 谷氨酸-C 在1:50 蛋白酶到基质蛋白比值 (w/w), 并在37°c 一夜之间以 1400 rpm 的搅动来孵化样品。</li>
	<li>酸化和淬火蛋白质消化样品通过添加甲酸到1% 的数量。</li>
	<li>
用亲水性-亲脂平衡固相萃取墨盒对样品进行脱盐。每个样品使用一个30毫克的吸附剂墨盒, 每个墨盒的最大5毫克材料为4,18。 注意: 在整个脱盐过程中, 保持墨盒内的吸附剂是很重要的。必要时, 关闭真空泵以减缓溶剂或样品通过墨盒的通过。<ol>
<li>将墨盒插入到端口 (s) 上的24端口玻璃块真空歧管, 并打开真空泵。保留未使用的端口的上限。如果有必要, 留下一个或两个端口, 在真空计上保持较低的压力。</li>
		<li>用800µL 80% 乙腈 (ACN)/0.2% 甲酸/19.8% 水将每个墨盒湿两次。确保溶剂水平保持2–3毫米以上的吸附剂水平。确保在流形上的真空计读数16.9–67.7 帕 (5–20汞)。</li>
		<li>平衡每盒三次, 800 µL 0.2% 甲酸在水中。确保在流形上的真空计读数16.9–67.7 帕 (5–20汞)。</li>
		<li>将多肽样本加载到墨盒上。确保歧管上的真空计读数6.77–8.47 帕 (2–2.5 汞), 流速不超过1毫升/分钟。</li>
		<li>用800µL 0.2% 甲酸在水中清洗每个墨盒三次。确保在流形上的真空计读数16.9–67.7 帕 (5–20汞)。</li>
		<li>从真空歧管中取出墨盒, 然后微细1.5 毫升。</li>
		<li>洗脱肽一次与800µL 80% ACN/0.2% 甲酸/19.8% 水, 并允许溶剂孵化与吸附剂为2–3分钟。</li>
		<li>使用吸管将溶剂推入墨盒内的吸附剂层。重复第二次洗脱400µL 80% ACN/0.2% 甲酸/19.8% 水成同1.5 毫升微细。</li>
	</ol>
</li>
	<li>用真空离心法干燥洗脱液 (固定离心率为 268 xg), 通常为 3 h。 注: 如有需要, 干燥的洗脱液样品可以储存在-20 摄氏度冷冻, 直到准备进行分馏。</li>
</ol>3. 分级肽使用离线碱性 pH 反相色谱法
<ol><li>重新暂停每酰和谷氨酸 C 的消化样品 (如1和2节所述) 在200µL 的缓冲 A (10 毫米甲酸铵在水中, pH 值 10), 在4摄氏度的涡流搅拌机上混合样品10分钟, 离心机为 17500 x g , 在室温下为10分钟。mperature。 注: 所生成的肽样本可以通过高 pH 值分离19,20离线分割。</li>
	<li>根据所使用的仪器和软件, 编制离线高效液相色谱分馏法。以下方法具体用于二进制液相色谱泵系统, 包括双波长紫外探测器、autosampler、分数收集器和 C18 柱 (4.6 毫米 x 250 毫米, 5 µm 粒度), 耐受 pH 值10。</li>
	<li>收集40个分数每分数2.1 分钟, 并结合每第四分数, 导致四最终池分数4。大量的聚集分数可用于进一步降低样本复杂度, 代价是质谱数据收集时间。</li>
	<li>干燥冻干机中的聚集馏分, 在水中将1毫升 50% ACN 和1% 甲酸中的干肽样品重新悬浮, 并转移到较小的样品容器中。通过真空离心 (固定离心率为 268 x g) 干燥转移的样品, 通常为 1 h。 注: 甲酸铵的低蒸气压可以使完全干燥困难。如果大量的甲酸铵盐在干燥后保持盐沉淀, 重复固相萃取。</li>
	<li>用索引保留时间 (µL) 肽标准混合物在水中重新悬浮 20 0.2% 甲酸的样品。 注: 样品现已准备好进行质谱分析。</li>
</ol>4. 分肽样品的直径分析
<ol><li>
用一个直径的 LC-ms (ms) 方法分析样品, 可以根据现有的质谱仪进行调整。用纳米 LC 2D 高效液相色谱系统在线连接到正交四极飞行时间 (QqTOF) 质谱仪进行了分析。
	<ol>
<li>程序的仪器软件, 使肽混合物转移到一个 C18 前柱芯片 (200 µm x 6 毫米 C18-CL 芯片, 3 µm, 300 Å) 和洗涤2µL/分钟10分钟与负载溶剂 (H2O/0.1% 甲酸) 脱盐。</li>
		<li>将多肽 chromatographically 在75µm x 15 厘米 C18-CL 芯片 (3 µm, 300 Å) 上, 以300个 h 梯度的流速, 使用典型的 (和 LC 系统特定的) 水和 ACN 溶剂缓冲2。</li>
		<li>生成可变窗口直径方法, 其中, 可变宽度的大范围窗口 (5 到 90 m/z) 从 Q1 四极的增量步骤传递到冲突单元格 (m/z 400-1, 250)。 注: 3.2 秒的循环时间包括 250 ms 前体离子扫描, 45 毫秒累积时间分别为64个带状段21,22。</li>
	</ol>
</li>
	<li>通过对样品的平行分析和光谱库的建立来识别多肽, 或者, 可以使用 PIQED 软件23直接从直径数据中识别多肽, 它处理数据处理的所有技术步骤,标识, 并导入到天际, 以便随后进行量化。</li>
</ol>5. 示例数据分析教程
<ol><li>
下载天际线软件 (http://proteome.gs.washington.edu/software/skyline) 并创建一个 PanoramaWeb 帐户 (https://panoramaweb.org)。 注: 有关使用数据的天际线描述的详细教程, 请参阅 (https://skyline.ms/wiki/home/software/Skyline/page.view？name=tutorials)。其他软件算法可用于从直径收购中提取光和重碎片离子峰值区域, 如打开的带状线24、将2.0 个插件插入 PeakView25和 Spectronaut26。<ol>
<li>从全景公共: https://panoramaweb.org/labkey/JoVEsuccBSA.url 下载琥珀酰化 BSA 代表结果(图 3) 的天际线数据文件。在全景网页上, 转到 "目标 MS 运行" 表, 下载天空. zip 文件选择右侧的下载链接。解压下载的. zip 文件夹, 双击地平线上的天空文件以在地平线上打开它。检查肽片段离子目标树 (左面板) 和四图谱从定义的百分比光琥珀酰化 (右面板)。</li>
		<li>选择 "查看" 菜单, 导航到 "峰值区域 |复制比较 "。包含条形图的 "峰值区域–复制比较" 面板将出现在窗口的右侧。在 "峰值区域" 面板中, 右键单击并选择 "转换 |单 ", 显示片段离子峰值区域为选定的片断离子。最后, 右键单击 "峰值区域" 面板并选择 "订单 |文件 "。</li>
		<li>在窗口左侧的目标树面板中, 右键单击肽 "E. LCKVASLRE。T |比率 |Oneheavy ", 它计算光离子信号在重离子信号, 轻/重的比率。 注: 这不等同于场地占用量的化学计量比 (重 + 轻)。</li>
		<li>请注意, 目标树现在报告光片段离子右侧的比值 L/小时。</li>
	</ol>
</li>
	<li>
确定含有所感兴趣的酰化部位的 "分化片段离子"。 注意: 如图 3所示, 具有这个确切的示例, 区分的离子被确定为 y7 (最高排列)、y8、b3和 b4。请注意, 非区分片段离子在目标树中显示的比率为1。<ol>
<li>在左面板目标树中, 在 588.30 + + LCKVASLRE 子节点的肽 E。T, 点击 y7 区分片段离子 K [y7 ]-902.49 + (rank1) [5] 注意到峰值高度和面积的增加。</li>
		<li>在左面板目标树中, 在 588.30 + + LCKVASLRE 子节点的肽 E。T, 单击 y5不区分片段离子A [y5 ]-575.31 + (秩 6) [1]。观察 y5片段离子图谱和峰值区域在所有样本的相同范围内保持不变。</li>
		<li>在目标树面板中, 浏览其他区分 (比 1) 和不区分 (比率为 1) 片段离子, 并注意到峰值区域条形图和图谱的变化和模式。</li>
		<li>确定区分离子的光和重对的精确峰值区域, 以计算化学计量 (为其他细节也看见迈尔et 。4). 点击 "查看 |文档网格 ", 文档网格将弹出。在文档网格的左上点, 单击 "视图 |过渡结果 "查看具有片段离子信息的多列表, 如肽序列、前体的规则、复制名称、等等
</li>
		<li>通过再次单击文档网格中的 "视图" 并选择 "编辑视图" 打开 "自定义视图" 窗口, 将报表格式更改为 "枢轴"。在 "自定义视图" 窗口的左下角, 检查 "数据透视复制名称" 并选择 "确定" 关闭 "自定义视图" 窗口。请注意, 现在 "文档网格: 转换结果" 表窗口重新排列, 以将复制名称、保留时间、区域、背景、峰值秩列 (1%、10%、50%、100% 琥珀酰化) 分组。</li>
		<li>为肽 "E. LCKVASLRE。T "在" 文档网格: 转换结果 "表中, 查找" 前向 "列、" 片段离子 "列和1pct_light_sw1 区域 (1% 琥珀酰化) 列。观察到该肽有两种前体离子, 即光 (前8行中的588.30 前驱体) 和重 (前8行中的 590.32)。</li>
		<li>在 "片段离子" 列中, 找到与光和重前驱离子对应的 y8 片段离子的两行。从1pct_light_sw1 区域列中的同一行中, 记录光 (15820) 的 y8 区域和重 (1426461)。</li>
	</ol>
</li>
	<li>使用这些峰值面积值, 计算琥珀酰化化学计量, 或修改的比例, 根据下面的公式, 并获得一个化学计量比率 0.01, 或1% 琥珀酰化, 这符合光琥珀酰化的比例, 在这个定义混合物: <img alt="Equation" src="/files/ftp_upload/57209/57209eq1.jpg">
</li>
	<li>用 y8 区分片段离子峰值面积值从10pct_light_sw1 区柱、144953 (光) 和 1188041 (重) 中计算10% 琥珀酰化的化学计量比, 以获得0.10 或10% 琥珀酰化的比值。</li>
	<li>用 y8 区分片段离子峰值面积值从100pct_light_sw1 区柱、954513 (光) 和 16407 (重) 中计算100% 琥珀酰化的化学计量比, 以获得0.98 或98% 琥珀酰化的比值。</li>
	<li>同样, 用 b3 区分片段离子峰值面积值从1pct_light_sw1 区域柱、55697 (光) 和 3149119 (重) 中计算1% 琥珀酰化的化学计量比, 以获得0.01 或1% 琥珀酰化的比值。</li>
	<li>使用所有区分片段离子 (y 和 b 离子) 的公式继续计算化学计量比率, 以获得1、10、50和100% 琥珀酰化混合物的赖氨酸琥珀酰化化学计量值。</li>
</ol>

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作者没有什么可透露的。

该协议提供了一种新的和准确的方法来量化站点特定的赖氨酸乙酰化和琥珀酰化占用, 可应用于整个蛋白质组。该方法依赖于测定内源性轻肽和外源重肽, 后者是通过氘醋酸酐-d6 和蛋白质的定量化学酰化,在体外生成的体内琥珀酸酸酐-d4 (图 1)。类似的方法使用了稳定的同位素标记本地未修改的赖氨酸残留物, 并根据前体离子-只有12或片段离子从 DDA 收购13,14进行场地占用量化。本协议适用于样品制备过程中的几个步骤, 以提高酰化效率, 并将化学标记扩展到琥珀酰化。使用直径采集的数据收集在整个可检测的质量范围内获得前体离子和 MS2 片段离子强度, 从而减少碎片离子干扰。通过在内部开发的天际线和自定义脚本进行分析和量化, 允许在一个站点上包含多个赖氨酸残渣和多个酰化物的多肽的现场占用量计算。
在本议定书的样本准备阶段, 应密切注意几个关键步骤。由于整个议定书都依赖于对所有赖氨酸残留物进行有效的化学修饰, 这一步骤至关重要。酸酐与游离胺反应, 因此必须避免在蛋白质裂解物中含有胺的缓冲液或污染物分子。此外, 在化学每酰化的步骤, 确保反应混合物的 ph 值调整回 ph 值8后, 每三孵化与酸酐试剂作为这种反应酸化混合物, 和 O 酰化的副作用可能形成。此外, 在消化前将8米含脲反应混合物稀释至约0.8 米尿素, 并检查 pH 值是否在最佳范围内, 对 endoproteinase 的最佳活性有重要意义。我们的协议的另一个关键部分是数据收集步骤和一个直径工作流的引入, 它克服了所有的 DDA 数据采样不一致, 并且允许在 MS2 片段离子水平上进行量化。直径测量方法的一个主要优点是在量化 MS1 前体离子信号 (正如以前的一些出版物所建议的) 时, 干扰的减少通常更容易和更成问题。
在编写高度复杂的示例时, 可以对工作流进行几项修改。在离线碱性 pH 反相 HPLC 分离后, 可以减少混合的分数, 以降低 LC/MS 所获得的汇集样品的复杂性. 此外, 还可以使用更长的色谱梯度在获取, 以允许更好的肽分离。另外, 多种蛋白酶可以用来消化样品, 以产生更多的裂解肽, 增加蛋白质赖氨酸残留物的覆盖率。试验运行和优化可以使用含有特定百分比的乙酰化或琥珀酰化的商业 BSA 进行。
对此协议的一个限制是, 由于在同一站点4中的其他赖氨酸修饰 (如甲基化或泛、等) 的未知, 可能会略微高估站点占用率。从光和重酰酰基修改的峰值面积计算, l/(升 H), 场地占用是根据假设, 在赖氨酸部位的总改性仅由内源 ' 光 ' 酰化 (l) 和化学标记 ' 重 '酰化 (H)。然而, 在一个站点体内的实际总酰化占用可能包括除乙酰化和/或琥珀酰化以外的其他修改。此外, 报告的同位素纯度的采购试剂醋酸酸酐-d6 (99%) 和琥珀酸酸酐-d4 (> 98%) 可能会导致小高估高达 2% (酰) 或 1% (乙酰) 的内源化酰化水平4。这些细微的高估在一起, 增加了在数量少于1% 的地点进行量化的困难。完整的化学酰肽和本地酰肽之间的巨大差异也会导致潜在的站点占用量化错误, 特别是对于低内源性酰肽27。最近的一项研究由 Weinert et al。27发现, 完整的每乙酰多肽之间的丰度差异会降低量化错误27。
使用此协议收集的化学计量 quantifications 可以确定特定蛋白质中重要的酰化化热点, 并形成生物学后续实验的假说, 例如某些感兴趣的地点定向诱变。该协议还可以揭示低酰化化学计量站的联合效应, 对蛋白质结构稳定性和局部细胞环境产生间接或细微的影响。此外, 本议定书的实施, 以测量乙酰化和琥珀酰化和其他可能的赖氨酸酰化修饰超越乙酰化, 将独特的洞察力的动态酰化作用的蛋白质在不同的生物条件。

NƐ-蛋白质赖氨酸残留的酰化是蛋白质功能的重要调节因子。赖氨酸乙酰化和其他 acylations, 如琥珀酰化, malonylation 和 glutarylation, 被认为是调节新陈代谢, 细胞信号, 和其他细胞进程1, 2, 3, 4, 并对代谢紊乱有影响5。许多研究发现赖氨酸酰修饰在哺乳动物组织和细胞系的不同条件下发生大的褶皱变化5,6,7,8以及细菌9,10,11然而, 这些相对褶皱的变化并没有提供对总蛋白修饰的比例的洞察力。研究报告的酰化站点占用的测量是稀缺的12,13,14, 尽管这些研究的相关性和需要, 因为它们提供更多的细节比相对褶皱变化。例如, 10 倍的变化可以表示站点占用增加从0.01 到 0.1%, 1 到 10%, 甚至10到100%。需要精确的化学计量测量来解释酰化的生物学意义, 并预测蛋白质结构的大小和可能的功能变化的影响。
以前的一种量化场地占用的方法是利用对未修饰赖氨酸残留物进行重稳定同位素化学标记, 然后用质谱法测定内源性 "光" 乙酰化与外源的比值,化学标记的 "重" 乙酰赖氨酸使用前体离子强度12。周et最近的另一项研究。13描述了一种类似的方法来评估赖氨酸乙酰化化学计量法, 它还使用了在稳定同位素标记的蛋白质中所有未经修饰的赖氨酸残留物的完全化学乙酰化, 但使用了片段离子强度, 以dda.Nakayasu et al。14使用了类似的 DDA 方法, 而是使用了轻和重乙酰赖氨酸 immonium 离子的比值进行量化。基于片段离子、immonium 或序列离子 (MS2) 的量化, 在大多数情况下, 与处理完整的肽前体离子 (MS1) 相比, 信号干扰更小。然而, 从 DDA 生成的 ms/ms 谱中对片段离子的量化可能会受到随机抽样缺陷的影响, 在这种情况下, 高丰度前体离子更可能被选用于 ms 或 ms, 从而导致有偏差和不完全的抽样前体离子。
这个新的工作流4 (图 1) 在概念上使用了一个稳定的同位素化学标记方法, 最初由巴埃萨et al开发。12, 但是工作流随后与直径耦合, 以收集在可检测的质量范围15、16提供精确的化学计量计算的前体和多片段离子丰度。
从含有酰化位点的片段离子的峰值区域, 或 ' 区分片段离子 ', 用于量化酰化的站点占用 (图 2)。不含修饰的片段离子具有相同的光和重的 m-/z 值, 用于肽的识别, 但不能用于定量。天际线软件17用于提取前体和片段峰值区域。如果在某些片段离子中检测到干扰, 则含有给定酰化点的多个片段离子的存在提供了灵活性。地平线上的数据可视化可以对轻-重碎片离子比率进行严格的检查。除了手动数据分析外, 还开发了一个内部的开源 R 包 (StiochiolyzeR (https://github.com/GibsonLab/StoichiolyzeR), 它使用了通过直径和量化的前体离子和片段离子数据。从含有多重赖氨酸残留物的多肽中的特定地点的酰化化学计量, 这一特性不可能与前体离子强度测量4。
该协议还演示了使用 endoproteinase 谷氨酸-c, 这是特定的 c 末端裂解在谷氨酸和天门冬氨酸残留, 而不是使用胰蛋白酶或 Arg C 蛋白酶, 因为后者往往产生非常大的和潜在的繁殖酰在酰赖氨酸残留物中阻断胰蛋白酶裂解而产生的蛋白水解肽。化学标签程序也扩大到包括使用琥珀酸酸酐 (图 1), 从而使琥珀酰化化学计量琥珀酰化的量化。除了对样品制备进行改进外, 采用离线、碱性 pH、反相 (bRP) 分离肽的方法进一步减少了量化干扰, 从而更好的消除卷积整个蛋白质组样品中的酰化化学计量学。这一方法的特点和突出几个优点: (1) 提高化学酰化的效率;(2) 除乙酰化外, 测定蛋白质琥珀酰化化学计量;(3) 提高了量化准确度。改进后的量化是由于从直径的片段离子信号中的干扰减少与 DDA 前体信号, 以及 bRP 的离线肽预分馏的实施。

<table><tbody><tr><td>aqueous acetic anhydride-&lt;em&gt;d&lt;sub&gt;6&lt;/sub&gt;&lt;/em&gt;</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>175641-1G</td><td>1% isotopic purity</td></tr><tr><td>succinic anhydride-&lt;em&gt;d&lt;sub&gt;4&lt;/sub&gt;&lt;/em&gt;</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>293741</td><td>2% isotopic purity</td></tr><tr><td>sequencing grade endoproteinase Glu-C</td><td>Roche, Indianapolis, IN, USA (through Sigma)</td><td>10791156001</td><td>1:50 protease to substrate protein ratio (wt:wt)</td></tr><tr><td>Oasis HLB Solid-Phase Extraction (SPE) cartridges</td><td>Waters Corp., Milford, MA, USA</td><td>WAT 094225</td><td></td></tr><tr><td>HPLC acetonitrile</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJAH015-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJAH365-4</td><td></td></tr><tr><td>iodoacetamide</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>I1149-25G</td><td></td></tr><tr><td>dithiothreitol (DTT)</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>D9779-5G</td><td></td></tr><tr><td>FLUKA formic acid (FA) for mass spec</td><td>Thomas Scientific, Swedesboro, NJ, USA</td><td>C988C27</td><td>~98%</td></tr><tr><td>urea</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>PI29700</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Pierce, Rockford, IL, USA</td><td>88341</td><td></td></tr><tr><td>triethylammonium bicarbonate (TEAB)</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>T7408-100ML</td><td></td></tr><tr><td>anhydrous dimethyl sulfoxide (DMSO)</td><td>Alfa Aesar, Tewksbury, MA, USA</td><td>43998</td><td></td></tr><tr><td>sodium hydroxide (NaOH)</td><td>EMDMillipore, Burlington, MA, USA</td><td>SX0590</td><td></td></tr><tr><td>50% hydroxylamine solution in water</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>438227-50ML</td><td></td></tr><tr><td>LC-MS grade water</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJLC365-4</td><td></td></tr><tr><td>LC-MS acetonitrile</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJLC015-4</td><td></td></tr><tr><td>Eppendorf Themomixer Compact</td><td>Eppendorf AG, Hamburg, Germany</td><td>T1317-1EA</td><td></td></tr><tr><td>Thermo Scientific Savant SPD131DDA SpeedVac Concentrator</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>SPD131DDA-115</td><td></td></tr><tr><td>Waters 1525 binary HPLC pump system</td><td>Waters Corp., Milford, MA, USA</td><td>WAT022939</td><td></td></tr><tr><td>Waters 2487 Dual Wavelength UV detector</td><td>Waters Corp., Milford, MA, USA</td><td>WAT081110</td><td></td></tr><tr><td>Waters 717plus Autosampler</td><td>Waters Corp., Milford, MA, USA</td><td>WAT022939</td><td></td></tr><tr><td>Waters Fraction Collector III</td><td>Waters Corp., Milford, MA, USA</td><td>186001878</td><td></td></tr><tr><td>Agilent Zorbax 300Extend C18 column</td><td>Agilent Technologies, Inc., Santa Clara, CA, USA</td><td>770995-902</td><td></td></tr><tr><td>Labconco Freeze Dry System Freezone 4.5 Lyophilizer</td><td>Labconco, Kansas City MO, USA</td><td>7751000</td><td></td></tr><tr><td>indexed retention time (iRT) normalization peptide standard</td><td>Biognosys AG, Schlieren, Zurich, Switzerland</td><td>Ki-3002-2</td><td></td></tr><tr><td>Ultra Plus nano-LC 2D HPLC system</td><td>Sciex LLC, Eksigent Division, Framingham, MA, USA</td><td>model# 845</td><td></td></tr><tr><td>orthogonal quadrupole time-of-flight (QqTOF) TripleTOF 5600 mass spectrometer</td><td>Sciex LLC, Framingham, MA, USA</td><td>per quote</td><td></td></tr><tr><td>orthogonal quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer</td><td>Sciex LLC, Framingham, MA, USA</td><td>per quote</td><td></td></tr><tr><td>C18 pre-column chip (200 &amp;mu;m &amp;times; 6 mm ChromXP C18-CL chip, 3 &amp;mu;m, 300 &amp;Aring;)</td><td>Sciex LLC, Framingham, MA, USA</td><td>804-00006</td><td></td></tr><tr><td>SWATH 2.0 plugin into PeakView 2.2</td><td>Sciex LLC, Framingham, MA, USA</td><td>software download Sciex</td><td></td></tr><tr><td>Spectronaut</td><td>Biognosys AG, Schlieren, Zurich, Switzerland</td><td>Sw-3001</td><td></td></tr><tr><td>C18-CL chip (75 &amp;micro;m x 15 cm ChromXP, 3 &amp;micro;m, 300 &amp;Aring;)</td><td>Sciex LLC, Framingham, MA, USA</td><td>804-00001</td><td></td></tr></tbody></table>

quantification of site-specific protein lysine acetylation and succinylation stoichiometry using data-independent acquisition mass spectrometry

蛋白质赖氨酸残留物的平移后修饰 (PTM) 由 NƐ-酰化诱导结构变化, 可以动态调节蛋白质的功能, 例如, 改变酶活性或调解相互作用。精确定量的地点特定的蛋白质酰化的占有, 或化学计量, 是理解的功能后果的全球低级别化学计量和个别高级别酰化化学计量的特定赖氨酸残留.其他组报告称, 通过比较内源性、自然丰酰化和外源的多肽前驱同位素的比值, 定量地引入了重同位素标记的酰化物, 测定了赖氨酸乙酰化化学计量方法。用稳定同位素标记的醋酸酸酐对蛋白质进行化学乙酰化。该协议描述了一种优化的方法, 其中包括: (1) 提高化学酰化效率, (2) 除了琥珀酰化, 还能测量蛋白质的含量, (3) 由于减少干扰, 使用碎片离子量化从数据无关的收购 (直径) 而不是前体离子信号的数据依赖获取 (DDA)。利用从片段离子中提取的峰值区域来进行量化, 也可以唯一地实现从含有多个赖氨酸残留物的蛋白水解肽中区分站点级酰化化学计量, 这是不可能使用前体离子量化信号。数据可视化在地平线上, 一个开源的定量蛋白质组学环境, 允许方便的数据检查和审查。这一工作流程共同提供了无偏、精确和准确的定量的站点特定赖氨酸乙酰化和琥珀酰化占用的整个蛋白质组, 这可能揭示和优先考虑生物学相关的酰化点。

在采集数据后, 从蛋白水解酰肽中测定 MS2 片段离子, 然后在地平线上提取离子图谱 (XIC), 并导出相应的光照和重峰区,最后用于计算场地占用。图 2A显示了前体离子 XIC 如何出现在光和重肽信号上的概念性说明,图 2B显示了相应的片段离子 XICs 与颜色编码 (红色 = 光; 蓝色 = 重) 用于区分含有赖氨酸酰化修饰的片段离子。
图 3显示了占用实验所产生的数据, 如上文所述分析了在1、10、50或100% 内部产生的光/重琥珀酰化 BSA 的预定义比率, 以及琥珀酰化占用量的确定。将直径占用数据导入天际线, 可以轻松地可视化目标树, 显示多肽序列, 以及用于光和重质前体离子的片段离子 (图 3A)。从每个已定义的琥珀酰化 BSA 比率的直径-MS 的数据揭示 immanently 的相对差异, 轻-重y 7 离子, 最高等级区分片段离子从确定的肽谱匹配 (用红色表示, 图 3B)。图 4显示了从 MS2 占用计算中得到的处理结果的概览, 它确认了输入蛋白质的琥珀酰化百分比占用, 这里包括琥珀酰化前 BSA。测定的赖氨酸琥珀酰化占20蛋白水解琥珀酰化肽获得的商业前琥珀酰化 BSA, 琥珀酰化在定义的百分比 (例如, 在 0, 1, 10 , 50和 100%) 显示在表2中。对于20蛋白水解 BSA 肽, 最高排列, 区分 MS2 片段离子被用来计算赖氨酸琥珀酰化 (Ksucc) 占用, 作为 L/(升 H)%。总的来说, 即使在低 stoichiometries, MS2-based 定量在测定酰化程度上也表现出很好的准确度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57209/57209fig1.jpg"> 图 1: 化学计量工作流.(A) 蛋白质用醋酸酐-d6 孵化三次, 以 acetylate 未修改的赖氨酸残留物。其次, 用 endoproteinase 谷氨酸 C 消化乙酰蛋白, 然后采用碱性-pH 反相色谱法 HPLC 分离水解肽。最后利用变前窗宽度的直径对多肽进行了分析。(B) 相同的工作流用于确定琥珀酰化化学计量, 如(A) 中所述, 但重酰化反应试剂改为琥珀酸酸酐-d4。图改编自迈耶et al。4 (j. 社会学课上质量 Spectrom, 打开选择)。<a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57209/57209fig2.jpg"> 图 2: 可能获得的数据和化学计量计算的示例.(A) 光 (L) 和重 (H) MS1 前体离子含有一乙酰赖氨酸, 其质量单位不同3。XICs 分别为红色和蓝色指示的光和重同位素的信封生成。(B) 从 MS2 片段离子 XICs 的量化, 可以使用 "区分" 光和重片段离子, 其中含有以红色和蓝色表示的乙酰化部位。常见片段离子以虚线黑色显示, 不包含修改的位置。图改编自迈耶et al。4 (j. 社会学课上质量 Spectrom, 打开选择)。<a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57209/57209fig3.jpg"> 图 3: 琥珀酰化蛋白水解 BSA 肽在不同的占有水平上的天际线可视化。(A) 地平线目标树, 显示蛋白水解肽 LCKsuccVASLRE 及其产生的片段离子。(B) 地平线提取的片段离子图谱在不同级别的琥珀酰化占用。最高级别的区分离子, y7, 在峰值区域1、10、50和100% 中增加, 这与琥珀酰化前 BSA (内部生成的) 的输入百分比相关。<a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57209/57209fig4.jpg"> 图 4: 评估琥珀酰化前 bsa 的 bsa 酰化化学计量。五种不同的 BSA 样本按规定的重度修改百分比 (e. g., 在0、1、10、50和 100%) 受到 MS2 的占用确定。20琥珀酰化 BSA 多肽的场地占用率是根据指定的光修改百分比(例如、0、1、10、50和 100%), 从最高排列的区分片段离子中测定出五种不同的 bsa 样品。盒晶须图显示20酰肽的分布, 从50% 多肽的值是在 ' 框 ' (25% 百分点到75% 百分点), 上部晶须指示值从75% 百分点到最大 (100%), 和较低的晶须表明从25% 位数到最小值 (0% 百分点)。(j. 社会学课上质量 Spectrom, 开放选择)。度量值的量化可以在表 2中找到。<a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>时间 (分钟)</td>
			<td>% A</td>
			<td>% B</td>
		</tr>
<tr>
<td>0.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
<tr>
<td>7.27</td>
			<td>92</td>
			<td>8</td>
		</tr>
<tr>
<td>45.27</td>
			<td>73</td>
			<td>27</td>
		</tr>
<tr>
<td>49.27</td>
			<td>69</td>
			<td>31</td>
		</tr>
<tr>
<td>65.27</td>
			<td>61</td>
			<td>39</td>
		</tr>
<tr>
<td>72.27</td>
			<td>40</td>
			<td>60</td>
		</tr>
<tr>
<td>80.00</td>
			<td>10</td>
			<td>90</td>
		</tr>
<tr>
<td>85.00</td>
			<td>10</td>
			<td>90</td>
		</tr>
<tr>
<td>86.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
<tr>
<td>120.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="2">流量: 0.7 毫升/分</td>
			<td></td>
		</tr>
<tr>
<td colspan="3">缓冲器 A:10 毫米甲酸铵在水中, pH 值10</td>
		</tr>
<tr>
<td colspan="3">缓冲 B:10 毫米甲酸铵在 90% ACN 和10% 水, pH 10</td>
		</tr>
<tr>
<td colspan="3">注: 两个流动相的 pH 值调整为10与整齐的氨</td>
		</tr>
</tbody></table>表 1: 脱机基本 pH 值反相 HPLC 分离的渐变.渐变长度: 120 分钟缓冲器 A:10 毫米甲酸铵在水中, pH 10。缓冲 B:10 毫米甲酸铵在 90% ACN 和10% 水, pH 10。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>l/升 + H 在%</td>
			<td>l/升 + H 在%</td>
			<td>l/升 + H 在%</td>
			<td>l/升 + H 在%</td>
			<td>l/升 + H 在%</td>
		</tr>
<tr>
<td>Ksucc 0%</td>
			<td>Ksucc 1%</td>
			<td>Ksucc 10%</td>
			<td>Ksucc 50%</td>
			<td>Ksucc 100%</td>
		</tr>
<tr>
<td>0。2</td>
			<td>1。7</td>
			<td>11。1</td>
			<td>50。9</td>
			<td>99。2</td>
		</tr>
<tr>
<td>1。2</td>
			<td>2。3</td>
			<td>12。3</td>
			<td>49。4</td>
			<td>97。6</td>
		</tr>
<tr>
<td>2。9</td>
			<td>3。8</td>
			<td>14</td>
			<td>48。6</td>
			<td>99。5</td>
		</tr>
<tr>
<td>0。5</td>
			<td>1。8</td>
			<td>11。7</td>
			<td>50。8</td>
			<td>99。5</td>
		</tr>
<tr>
<td>0。2</td>
			<td>1。7</td>
			<td>11。1</td>
			<td>48。3</td>
			<td>98。2</td>
		</tr>
<tr>
<td>0。2</td>
			<td>1。2</td>
			<td>11</td>
			<td>47。5</td>
			<td>96。1</td>
		</tr>
<tr>
<td>0。3</td>
			<td>1。6</td>
			<td>12。8</td>
			<td>51</td>
			<td>99。2</td>
		</tr>
<tr>
<td>3。7</td>
			<td>5。2</td>
			<td>14。7</td>
			<td>51。9</td>
			<td>89。5</td>
		</tr>
<tr>
<td>1。5</td>
			<td>1。8</td>
			<td>12。5</td>
			<td>47。2</td>
			<td>91。1</td>
		</tr>
<tr>
<td>0。8</td>
			<td>1。5</td>
			<td>11。3</td>
			<td>48。4</td>
			<td>96。8</td>
		</tr>
<tr>
<td>0。2</td>
			<td>1。6</td>
			<td>13。9</td>
			<td>49。7</td>
			<td>98。9</td>
		</tr>
<tr>
<td>0。1</td>
			<td>1。1</td>
			<td>10。7</td>
			<td>48。2</td>
			<td>98。6</td>
		</tr>
<tr>
<td>0。2</td>
			<td>0。9</td>
			<td>10。3</td>
			<td>49。4</td>
			<td>99。4</td>
		</tr>
<tr>
<td>0。5</td>
			<td>2。5</td>
			<td>17。2</td>
			<td>52。3</td>
			<td>97。5</td>
		</tr>
<tr>
<td>0。1</td>
			<td>3。5</td>
			<td>20。8</td>
			<td>51</td>
			<td>99</td>
		</tr>
<tr>
<td>0。3</td>
			<td>1。9</td>
			<td>10。7</td>
			<td>49。6</td>
			<td>98。2</td>
		</tr>
<tr>
<td>2</td>
			<td>1。7</td>
			<td>10。9</td>
			<td>47。7</td>
			<td>96。3</td>
		</tr>
<tr>
<td>0。3</td>
			<td>1。2</td>
			<td>10</td>
			<td>53</td>
			<td>98</td>
		</tr>
<tr>
<td>1。1</td>
			<td>1。8</td>
			<td>12。9</td>
			<td>57。9</td>
			<td>94。1</td>
		</tr>
<tr>
<td>0。2</td>
			<td>1。4</td>
			<td>11。6</td>
			<td>48。6</td>
			<td>98。8</td>
		</tr>
</tbody></table>表 2: 对20蛋白水解琥珀酰化肽测定的 BSA 赖氨酸琥珀酰化占用量进行量化。琥珀酰化肽从商业前琥珀酰化 BSA 获得, 琥珀酰化在定义的百分比 (例如, 在 0, 1, 10, 50 和 100%)。对于20蛋白水解 BSA 肽, 最高排列, 区分 MS2 片段离子被用来计算赖氨酸琥珀酰化 (Ksucc) 占用, 作为 L/(升 H)%。

Watch this Scientific Journal Video about Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry at JoVE.com

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

在这里, 我们通过对在定量之后引入的修改的比例分析, 对整个蛋白质组的站点特异性蛋白质乙酰化和/或琥珀酰化占用 (化学计量) 进行无偏量化。用稳定同位素标记的酸酐化学酰化反应。结合敏感数据无关的采集质谱, 得到了准确的场地占用量测量。

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

quantification-site-specific-protein-lysine-acetylation-succinylation

Research

Education

JoVE Core

JoVE Journal

Organic Chemistry

Biology

Unit 1

Amines

SCIENTISTS IN ACTION

11.5K 次观看.  Buck Institute for Research on Aging. 在这里, 我们通过对在定量之后引入的修改的比例分析, 对整个蛋白质组的站点特异性蛋白质乙酰化和/或琥珀酰化占用 (化学计量) 进行无偏量化。用稳定同位素标记的酸酐化学酰化反应。结合敏感数据无关的采集质谱, 得到了准确的场地占用量测量。

视频: Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

科研

化学

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

生物化学

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

医学

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

使用阿基-树脂辅助捕获检测蛋白质S-囊肿

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

免疫与感染

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

生物学

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法:从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...