Name: two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome
Uploaded: 2018-04-02T13:00:00.000+00:00
Duration: 12 min 34 s
Description: Watch this Scientific Journal Video about Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome at JoVE.com

这项工作得到中国国家自然科学基金 (81572278 和81272798对 xz) 的支持, 中国 "863" 计划项目 (2014AA020610-1)、象牙医院人才引进基金 (xz) 和湖南中国省级自然科学基金 (授予14JJ7008 号)。作者还承认 Desiderio 博士在田纳西大学健康科学中心的科学贡献。X.Z. 连续开发和使用2种方法从2001年起对垂体腺瘤蛋白质组进行分析, 构思了本手稿的概念, 撰写和修订了手稿, 协调了相关工作, 并负责财务支持及相应工作。Y.L. 参加了手稿的修订。他参加了参考资料的收集和手稿的修订。所有作者都批准了最后的手稿。

本议定书遵循中国中南大学象牙医院医学伦理学委员会的指导方针。在整个实验过程中应佩戴头帽和手套, 以避免皮肤和毛发中的角蛋白污染8。
1. 样品的制备
<ol><li>从神经外科部门收集 PA 组织 (0.2-0.5 毫克)。立即冷冻液氮, 然后转移到-80 °c 存放。</li>
	<li>在去离子蒸馏水中加入2毫升0.9% 氯化钠 (ddH2O)。使用此解决方案可以轻松地从组织表面 (3x) 中冲洗血液。 注意: ddH2O 的电导率为 18.2 MΩ/厘米, 在整个协议中使用。有些 tissuemight 在从组织表面清洗血液时会丢失。</li>
	<li>添加体积 (10 毫升; 4 °c) 溶液中含有2米醋酸和 0.1% (v/v) β-巯基乙醇每 0.5-0.6 克组织, 融汇 (1 分钟, 1.3万 rpm, 4 °c, 10x) 与组织均质体, 油脂实验匀浆二十年代, lyophilize, 并存储在−80°c。</li>
	<li>采用二辛可宁酸测定试剂盒测定冻干、匀质样品的蛋白质含量。 注意:衡量标准的量化不是绝对量化的, 测量结果将被不同的技术员和实验代理人改变。在不同的实验中应采用固定浓度样本标准。因此, 在预设计的实验中, 每2D 凝胶的蛋白质的最终加载量将与银染色或考马斯染色的好分辨率图像一起确定。<ol>
<li>添加约300µg 的冻干均匀样品到一个体积 (282 µL) 的蛋白质提取缓冲 (8 米尿素和4% 章), 其次是站在 2 h, sonicating 在水浴为5分钟, 旋转 1 h, sonicating 再次在水浴为5分钟, 再次旋转1小时, 离心在 1.5万 x g 处为20分钟。</li>
		<li>制备 bsa 标准溶液的浓度如前所述: 0, 25, 125, 250, 500, 750, 1500, 2000 µg/毫升与商业 BSA 标准 (2 毫克/毫升)。</li>
		<li>在使用前, 将可调试剂 A 和 b (a: b = 50:1) 作为可供选择的工作解决方案。</li>
		<li>在离心管中加入0.1 毫升的样品或标准溶液至2毫升的可工作溶液, 然后混合然后在37摄氏度处孵化30分钟。最后在室温下冷却10分钟, 并测量562 nm的外径值。</li>
		<li>计算标准线性线 (562 nm与 BSA 浓度), 以获得用于计算具有562nm值的样本蛋白质含量的回归方程。</li>
	</ol>
</li>
	<li>使用150µg 的蛋白质当量冻干样品进行银染色, 或500µg 蛋白质考马斯染色, 18 厘米固定 ph 梯度 (IPG) 条 ph 值3-10 非线性 (NL)。 注意:IPG 干带是0.5 毫米厚和3毫米宽, 不同的长度, 包括 7, 11, 13, 18 和24厘米, 和不同的 ph 值范围, 包括 ph 值 4-5, 4-7, 6-9, 和3-10 在线性或非线性 ph 梯度。所使用的 IPG 缓冲区必须适合条形27、28。</li>
	<li>1.6.Add 250 µL 提取缓冲器 (7 米尿素, 2 米硫脲, 4% (瓦特/v) 章, 100 毫米 (使用前添加), 0.5% 伏/v pharmalyte (使用前添加), 和溴酚蓝色的痕迹)。</li>
	<li>将溶液保持在30摄氏度以下, 后跟涡流5分钟, sonicating 5 分钟, 旋转50分钟。</li>
	<li>添加110µL 的补液缓冲 (7 米尿素, 2 米硫脲, 4% (瓦特/v) 章, 60 毫米 (使用前添加), 0.5% 伏/v IPG 缓冲 (使用前添加), 和溴酚蓝色的踪迹)。油脂实验5分钟。然后旋转样品50分钟, 涡流为5分钟, 离心机为20分钟在 1.5万 x g。</li>
	<li>收集上清 (350 µL) 作为蛋白质样品溶液8。</li>
</ol>2. 二维凝胶电泳
<ol><li>IEF (第一维度): 在等电聚焦系统上执行 IEF, 如下所述。
	<ol>
<li>在18厘米 IPG 带支架的插槽中添加350µL 的蛋白质样品溶液。</li>
		<li>把一个18厘米 IPG 条凝胶-侧下到蛋白质样品溶液 (避免气泡)。</li>
		<li>添加3-4 毫升矿物油覆盖 IPG 条。</li>
		<li>将 IPG 带支架装配到系统背面 (+) 板上的尖端和前端 (−) 板上的方端上的等电聚焦。</li>
		<li>水化过夜 (室温下18小时)。</li>
		<li>设置 IEF 参数: 最大电流30µA 每条, 20 °c;250伏, 1 小时, 125 Vh, 台阶和保持;1000 V, 1 小时, 500 Vh, 梯度;8000 V, 1 小时, 4000 Vh, 梯度;8000伏, 4 小时, 3.2万 Vh, 台阶和保持;和 500 V, 0.5 小时, 250 Vh, 步骤和举行。让它跑到总时间7.5 小时和 36875 Vh。</li>
		<li>IEF 后, 取出每个 IPG 条, 用不溶解的纸巾取出多余的矿物油。现在把小条包装在一张塑料包装纸里, 并存放在−80°c。 注意:IPGstrip 持有人应使用 IPGstrip 清洁液和蒸馏水清洗。</li>
	</ol></li>
	<li>sds 页 (第二维度): 在垂直细胞电泳系统中执行 sds 页面 注意:每个 SDS 页面的凝胶尺寸应为 255 x 190 x 1 毫米。<ol>
<li>使用多凝胶脚轮铸造12% 页解决凝胶。对于铸件12% 页凝胶, 请执行以下步骤。<ol>
<li>添加270毫升的 ddH2O, 150 毫升1.5 米三盐酸 (pH 8.8), 180 毫升 40% (瓦特/v) 丙烯酰胺/bisacrylamide 溶液 (29:1, 40% 瓦特/v 丙烯酰胺和1.38% 瓦特/v n '-methylenebisacrylamide), 3 毫升的10% 硫酸铵, 和150µL TEMED, 使凝胶在烧杯中的解决方案。轻轻搅拌, 避免气泡。</li>
			<li>将凝胶溶液轻轻倒入多播室, 达到预期凝胶高度 (19 厘米)。</li>
			<li>立即在每个解析凝胶上添加大约3毫升 ddH2O 以覆盖凝胶。让凝胶聚合约1小时。</li>
		</ol>
</li>
		<li>准备 20 L 的电泳缓冲器 (25 毫米三, 192 毫米甘油, 0.1% SDS)。用这个缓冲器填充电泳分离单元缓冲罐。</li>
		<li>从冰箱里取出重点的 IPG 条并平衡10分钟, 通过轻轻摇摆在4毫升减少平衡缓冲 (375 毫米三 HCl (pH 8.8), 6 M 尿素, 2% (w/v) SDS, 20% (v/v) 甘油, 2% 瓦特/v (添加之前使用), 溴酚蓝色的痕迹)。</li>
		<li>平衡4毫升烷基化平衡缓冲器 (375 毫米三盐酸 (pH 为 8.8), 轻轻摇动减少的 IPG 条, 以10分钟, 6 米尿素, 2% (瓦特/v) SDS, 20% (v/v) 甘油, 2.5% 瓦特/v iodoacetamide (使用前添加), 和溴酚蓝色的痕迹)。</li>
		<li>在凝胶平衡时, 拆开多播室, 取出准备好的凝胶盒。用 ddH2冲洗 3x, 用不溶解的纸巾去除多余的 ddH2o, 然后放入一个凝胶架。</li>
		<li>用电泳缓冲器冲洗平衡 IPG 条, 用不溶性纸巾去除 IPG 条表面的过量液体。</li>
		<li>把一个 IPG 带到解决 SDS 页凝胶, 并让 IPG 条的塑料侧接触较长的玻璃板和尖端向左。</li>
		<li>将3-4 毫升的热1% 琼脂糖在 sds 电泳缓冲器 (~ 80 °c) 快速地密封在每个 sds 页凝胶顶部的 IPG 条, 并将 IPG 条的顶部放到较短的玻璃板的顶部没有气泡, 然后聚合10分钟。</li>
		<li>在垂直电泳系统中, 在塑料垫片之间垂直插入组装的胶盒。把凝胶的顶部与 IPG 条旁边的阴极 (−)。</li>
		<li>调整电泳缓冲液的水平, 浸泡凝胶盒。</li>
		<li>在恒定电压模式下连接并设置电源/控制单元, 并在监测染料时, 在200伏处运行约370分钟。</li>
		<li>运行后, 取出电泳系统中的胶盒, 轻轻地从胶盒中取出凝胶, 避免撕裂凝胶, 然后染色2分离蛋白。</li>
	</ol></li>
	<li>2种脱分离蛋白的染色
	<ol>
<li>
银色染色
		<ol>
<li>将两只手放在含有250毫升固定溶液 (50% 甲醇 (v/v) 和 5% (v/v) 乙酸) 的托盘中, 轻轻地摇动凝胶20分钟。</li>
			<li>丢弃溶液, 在托盘中加入250毫升 50% (v/v) 甲醇, 轻轻摇动10分钟。</li>
			<li>丢弃甲醇并在托盘中添加250毫升的 ddH2O。轻轻摇动10分钟。</li>
			<li>在250毫升的感光缓冲器中轻轻摇动1分钟的凝胶, 含 0.02% (瓦特/v) 硫代硫酸钠, 并丢弃液体。</li>
			<li>在250毫升的 ddH2O 中轻轻摇动凝胶1分钟 (重复一次)。每一步后都要丢弃液体。</li>
			<li>在250毫升银反应溶液中轻轻摇动凝胶20分钟 (0.1% (瓦特/v) 硝酸银与200µL 37% (v/v) 在使用前添加甲醛。</li>
			<li>在250毫升的 ddH2O 中轻轻摇动凝胶1分钟 (重复一次)。每一步后都要丢弃液体。</li>
			<li>在250毫升开发溶液 (3% (瓦特/v) 碳酸钠与100µL 37% (v/v) 甲醛在使用之前添加) 轻轻摇动凝胶直到获得所需的染色强度 (通常为1-3 分钟), 然后丢弃液体。</li>
			<li>在250毫升停止溶液 (5% (v/v) 醋酸) 中轻轻摇动10分钟的凝胶, 并丢弃液体。</li>
			<li>在250毫升的 ddH2O 中轻轻摇动5分钟, 然后丢弃液体。</li>
			<li>保持凝胶在250毫升储存溶液的8.8% 甘油在4摄氏度。</li>
		</ol>
</li>
		<li>
考马斯亮蓝染色
		<ol>
<li>在 ddH2O 中溶解考马斯亮蓝 G250 0.3 克、硫酸铵25克和磷酸25毫升, 并多达200毫升的总容积, 制备考马斯染色溶液。使用前, 加入甲醇50毫升。</li>
			<li>在凝胶上加入250毫升的考马斯染色液, 在室温下轻轻摇动直到出现明显的蛋白斑点 (~ 2-4 小时)。扔掉液体。</li>
			<li>添加250毫升的 ddH2O, 并缓慢摇动5分钟, 然后再两次。扔掉液体。</li>
			<li>加入250毫升的脱色溶液 (20% (v/v) 乙醇), 慢慢摇动, 直到背景色变得几乎无色, 并丢弃液体。</li>
			<li>更换新的脱色解决方案时, 旧的是天黑了, 并丢弃液体。</li>
			<li>添加250毫升的 ddH2O 并缓慢摇动5分钟. 丢弃液体。</li>
			<li>添加250毫升的存储溶液 (8.8% 甘油), 并保持在4摄氏度。</li>
		</ol>
</li>
	</ol></li>
	<li>二维凝胶电泳图像分析
	<ol>
<li>用平板扫描仪将染色的凝胶数字化。</li>
		<li>将图像输入2D 凝胶图像分析系统。</li>
		<li>根据制造商的协议, 使用2D 凝胶图像分析软件检测和量化每个斑点。</li>
		<li>匹配不同凝胶之间的斑点。</li>
	</ol></li>
</ol>3. 用 MS 鉴别2种分离蛋白
<ol><li>胰蛋白酶肽合剂的制备 注意:渗或低保留管和吸管提示应用于避免任何蛋白质或肽的损失。<ol>
<li>
银凝胶脱色
		<ol>
<li>将凝胶点放到1.5 毫升渗管中, 并在500µL ddH2O 6 次洗涤。</li>
			<li>将洗净的凝胶放入新的1.5 毫升渗管中, 并将其切成许多小块 (0.5-1 毫米3)。</li>
			<li>Destain 的20µL 的凝胶件, 混合30毫米钾氰化钾的一部分和100毫米硫酸钠 (1:1) 的一部分, 在使用之前, 让褐色的颜色消失 (通常1-2 分钟)。扔掉液体。</li>
			<li>用20µL ddH2O 5-6 次清洗凝胶件, 让黄色颜色消失。每一步后都要丢弃液体。</li>
			<li>添加20µL 200 毫米碳酸氢铵的凝胶件, 并保持20分钟. 用 ddH2O (20 µL, 1 次) 清洗凝胶件。扔掉液体。</li>
			<li>用30µL 乙腈至少两次脱水凝胶件, 使凝胶件变成不透明的白色, 真空离心机干燥30分钟。</li>
		</ol>
</li>
		<li>
考马斯亮蓝凝胶脱色
		<ol>
<li>将凝胶点切成1.5 毫升的渗管, 并与500µL ddH2O 至少3次清洗。</li>
			<li>将洗涤后的凝胶放入新的1.5 毫升渗管中, 用吸管尖将其切成几片 (0.5-1 毫米3)。</li>
			<li>Destain 凝胶在50-100 µL 脱色溶液 (1:1 v/v 混合200毫米 NH4HCO3与乙腈) 取决于胶量, 孵化在37°c 在水浴。重复并添加新鲜的脱色解决方案, 直到颜色消失 (~ 1 小时)。每一步后都要丢弃液体。</li>
			<li>用20µL ddH2O 清洗凝胶件一次。</li>
			<li>将凝胶片脱水100µL 乙腈 (至少两次), 使凝胶件变成不透明的白色。</li>
			<li>真空干燥30分钟。</li>
		</ol>
</li>
		<li>
凝胶片中蛋白质的内胶胰蛋白酶消化
		<ol>
<li>将20µg (833 pmol) 冻干胰蛋白酶粉溶于50毫米醋酸100µL。</li>
			<li>将胰蛋白酶溶液稀释20µL (200 µL) 至 16 ng/µL, 230 µL 50 毫米碳酸氢铵。</li>
			<li>添加20-30 µL (0.32-0. 48 µg) 的稀释胰蛋白酶溶液, 每管含有干胶件。在37摄氏度的水浴中孵育18-20 小时。让溶液站在4摄氏度30分钟。</li>
			<li>十年代温和离心解决方案. 油脂实验在水浴中5-6 分钟, 在30摄氏度, 然后离心机在 1.2万 x g 2 分钟。</li>
			<li>将含有胰蛋白酶肽混合物的上清液收集成0.5 毫升的渗管。</li>
		</ol>
</li>
		<li>
C18 柱纯化胰蛋白酶肽合剂。
		<ol>
<li>用乙腈 (10 µL, 5 次), 然后用50% 乙腈 (10 µL, 5 次) 清洗每个 C18 柱。</li>
			<li>平衡与0.1% 氟乙酸 (TFA) (10 µL, 5 倍)。</li>
			<li>将样品通过准备好的 C18 柱向上和向下吸管15次。</li>
			<li>用10µL 0.1% TFA 清洗样品2x。 注: 纯化的胰蛋白酶肽保留在 C18 柱中。</li>
			<li>添加一个体积 (6 µL) 溶液 (85% v/v 乙腈/0.1% v/v 甲酸) 在一个干净的0.5 毫升渗管, 吸管这个溶液轻轻和缓慢向上和向下通过 peptide-C18 珠 10x, 以洗涤的债券多肽到溶液中, 风干, 并存储 (−20°c) 进行 MS 分析。</li>
		</ol>
</li>
	</ol></li>
	<li>MS 分析
	<ol>
<li>
MALDI-MS PMF 分析
		<ol>
<li>添加4µL 85% v/v 乙腈/0.1% 伏/v TFA, 以溶解纯化的胰蛋白酶肽混合物。将2µL 溶解胰蛋白酶肽混合物放入 MALDI 板上, 并立即加入2µL 一个氰基-4-肉桂酸 (CHCA) 溶液, 其含10克/升的500毫升/升乙腈1毫升/升 TFA, 然后风干。</li>
			<li>将胰蛋白酶肽混合物的 MALDI 板加载到 MALDI 质谱仪中。 注意:仪器参数设置为反射器模式, 具有 20 kV 加速电压、正极性和 160 ns 延时提取时间。每一个 MS 频谱是获得200激光射击与范围的 m/z 1000 到4000后, 外部校准与标准肽质量。</li>
			<li>使用 ms 频谱处理软件处理每个 MS 频谱, 包括基线校正、噪声去除 (5%) 和峰值 deisotoping。</li>
			<li>在空白凝胶实验的并行处理中, 从胰蛋白酶、基质、角质和其他未知污染物中去除污染的大量物质, 纠正各 MS 光谱中的质量清单。</li>
			<li>将已更正的质量列表输入到基于 PMF 的蛋白质搜索软件中, 以识别瑞士-波特蛋白数据库中的蛋白质, 其搜索参数包括 PMF 搜索类型、人、胰蛋白酶与1最大漏卵裂、固定carbamidomethyl 半胱氨酸, 变蛋氨酸氧化, monoisotopic, 肽质量耐受 50 ppm, 肽充电状态 (1 +), 和信号到噪声 (S/N) 5.0。</li>
			<li>识别具有统计学意义的蛋白质分数的蛋白质, 蛋白质评分 =-10 x 对数 (p), 其中 p 是观察到的匹配是随机事件的概率。</li>
		</ol>
</li>
		<li>
MALDI-ms 分析
		<ol>
<li>添加4µL 50% v/v 乙腈/0.1% 伏/v TFA, 以溶解纯化的胰蛋白酶肽混合物。每个纯化的胰蛋白酶肽混合物 (0.5 µL) 被发现在一个384井 MALDI 板上, 立即发现0.5 µL 饱和 CHCA 基质在50% 乙腈/0.1% TFA 上的肽样品, 然后干燥在空气中。</li>
			<li>让 MALDI 的毫秒/ms 自动管理, 使用100激光拍摄获得一个 MS1 频谱, 并积累6重复 MS1 光谱成一个合成 MS1 频谱。</li>
			<li>从每种合成 MS1 谱中设置4个最强的前体离子, 用于 ms 和 ms 分析。使用100激光射出每一个前体离子获得一个 ms/ms 频谱, 并积累4重复 ms/毫秒谱成一个合成 ms/ms 频谱。</li>
			<li>使用合成 ms/ms 数据通过基于 ms/ms 的蛋白质搜索软件搜索蛋白质, 其中包括 ms/ms 离子搜索、瑞士-波特人类数据库、最多1个漏卵裂的胰蛋白酶、固定 carbamidomethyl (C)、变量氧化 (M), monoisotopic 质量值, 肽质量耐受 100 ppm, 和片段质量耐受 0.7 Da。</li>
			<li>根据 Mowse 分数确定具有统计学意义的概率的蛋白质, 离子分数 =-10 x 对数 (p), 其中 p 是观察到的匹配是随机事件的概率。</li>
		</ol>
</li>
		<li>
LC-ms 分析
		<ol>
<li>添加6µL 5% v/v 乙腈/0.1% v/v 甲酸, 以溶解纯化的胰蛋白酶肽混合物。</li>
			<li>采用高效液相色谱 (HPLC) 系统与电喷雾电离 (ESI)-ms/ms 质谱仪进行在线耦合, 采用 ms/ms 数据获取管理软件来管理整个操作。</li>
			<li>使用 C18 陷印柱 (300 µm 编号 x 5 毫米长度), 和反相 C18 柱 (75 µm id x 15 厘米长度) 与分离梯度在98% 溶剂 a (0.1% 甲酸) 和2% 溶剂 B (0.1% 甲酸在100% 乙腈) 为2分钟, 2% 到40% 溶剂 b 为45分钟, 40% 到95% 溶剂 b 为5分钟, 95% 溶剂 b 为10分钟 (总共65分钟 300 nL/分钟)。</li>
			<li>设置正离子模式和依赖数据的自动测量模式获取 ms 和 ms/ms 谱, 并设置碰撞诱导离解 (CID) 离子碎片。</li>
			<li>将每个 MS 扫描设置为 m/z 350-1 的质量范围, 800 的分辨率为 10万, 在m/z 400 中。对 MS 扫描中的7最强离子进行扫描。</li>
			<li>使用 ms/ms 为基础的蛋白质搜索软件 I 搜索数据库, 以识别蛋白质。<ol>
<li>选择人类蛋白质数据库。</li>
				<li>选择胰蛋白酶与一个错过的分裂网站允许。</li>
				<li>设置肽耐受性 (±10 ppm), 片段离子耐受 (±0.8 Da), 肽电荷 (2 +, 3 + 和 4 +), 固定 carbamidomethylation 在半胱氨酸, 并在蛋氨酸的可变氧化。</li>
				<li>设置错误发现率 (罗斯福) < 1%, 并且只接受等级为1的多肽。</li>
				<li>用 PSM≥1识别蛋白质 (PSMs: 蛋白质的已识别肽序列 (PSMs) 总数, 包括重复识别的) 和每种蛋白质至少两个独特的肽。</li>
			</ol>
</li>
			<li>另外, 使用 ms/ms 为基础的蛋白质搜索软件 II 搜索数据库, 以识别蛋白质。<ol>
<li>将原始 MS 文件转换为. mgf 文件。</li>
				<li>选择了人类蛋白质数据库 (uniprothuman_20161031 fasta, 其中包含70940个序列和23897047个残留物)。</li>
				<li>选择胰蛋白酶消化, 最大为2错过分裂。</li>
				<li>设置固定 carbamidomethyl (C), 可变 deamidated (NQ) 和氧化 (M), 前体离子质量耐受 10 ppm, 女儿离子质量耐受 0.8 Da, 和 monoisotopic 峰。</li>
				<li>确定具有显著阈值0.05 和至少2个独特肽的蛋白质。</li>
			</ol>
</li>
		</ol>
</li>
	</ol></li>
</ol>

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作者声明, 没有利益冲突。

2DE, 再加上 ms 方法, 包括 2 PMF ms 和2的 ms/毫秒, 已成功地应用于我们的长期计划-使用蛋白质组学研究人类的消防协会蛋白质的变化和分子网络的变化, 以阐明分子机制和NFPAs 的有效生物标志物的发现。2基于 de 的比较蛋白质组学具有良好的重现性, 在鉴定消防协会蛋白质多样性方面起着重要作用9,10,11,12<...

PA 是一种常见的肿瘤, 发生在人类垂体腺的下丘脑-垂体靶向系统, 这在人类内分泌系统中扮演重要的角色。PA 包括临床功能和非功能性 PAs (平安险和消防协会)1,2。在早期诊断和治疗方面, 消防协会是困难的, 因为只有轻微升高的荷尔蒙水平 (例如, LH, 和 FSH) 的血液比平安险, 这大大提高了血液中相应荷尔蒙的水平3,4 ,5。分子机制的澄清和有效生物标志物的发现对消防协会的诊断、治疗和预后具有重要的临床意义。我们的长期目标是开发和使用蛋白质组学方法研究消防协会, 以发现可靠的生物标志物, 以澄清其分子机制, 并确认有效的治疗目标, 以及诊断和预后标志。2-DE 结合 MS 方法已广泛应用于我们的长期研究计划, 关于人类 PA 蛋白质组1,2,6,7, 包括建立蛋白质组参考映射3,8, 差异表达的蛋白质剖面分析9,10,11,12,13, 荷尔蒙变体14 ,15, 平移后修改, 如磷酸化14和酪氨酸硝化16,17,18, 入侵相对的蛋白质组变异无创 NFPAs19和消防协会亚型13的蛋白质组异质性, 导致了多种重要通路网络 (线粒体功能障碍、细胞周期失调、氧化应激和 MAPK 信号) 的发现。系统异常) 在消防协会13,19,20中更改。
2DE. 根据它们的等电点 (pI) (电聚焦, IEF) 和分子量 (通过月桂酸钠聚丙烯酰胺凝胶电泳, SDS 页)1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. 这是蛋白质组学领域的一种常见的和经典的分离技术, 自从 1995年24中引入了蛋白质组和蛋白质的概念。ms 是找出2种分离蛋白的关键技术, 包括 PMF 和 ms/ms 策略。MS 仪器的飞速发展, 特别是在检测灵敏度和分辨率方面, 结合 LC 系统的改进, 大大提高了蛋白质组中低或极低丰度蛋白质的识别率, 最大限度地蛋白质组的覆盖范围。它还挑战我们的传统观念, 在分析复杂的人体组织蛋白质组中只有一个或两个蛋白质存在于一个2D 凝胶点, 并提供了一个机会, 以确定在一个2D 凝胶点的多蛋白在复杂的人体组织分析蛋白质组, 并最大限度地覆盖了消防协会蛋白质组。
在这里, 我们描述了 2 MALDI ms PMF、2 MALDI ms/毫秒和2个非 LC ms/毫秒的详细协议, 这些方法已成功地用于人体消防协会蛋白质组的分析。这些协议包括: 样品的制备, 第一维 (电聚焦, IEF), 第二维 (SDS 页), 蛋白质的可视化 (银染色和考马斯蓝染色), 2D 凝胶的图像分析, 凝胶胰蛋白酶消化,纯化胰蛋白酶肽、PMF、ms/ms 和数据库灼热的3,8,25,26。此外, 该协议容易转化为分析其他人体组织蛋白质组。

<table><tbody><tr><td>Ettan IPGphor 3&amp;nbsp;</td><td>GE Healthcare</td><td></td><td>isoelectric focusing system.</td></tr><tr><td>Ettan DALTsix multigel caster</td><td>Amersham Pharmacia Biotech, Piscataway, NJ, USA</td><td></td><td></td></tr><tr><td>Ettan DALT II System</td><td>Amersham Pharmacia Biotech, Piscataway, NJ, USA</td><td></td><td>The vertical electrophoresis system</td></tr><tr><td>Ettan IPGphor strip holder</td><td>Amersham Pharmacia Biotech, Piscataway, NJ, USA</td><td></td><td></td></tr><tr><td>Ettan DALTsix multigel caster</td><td>Amersham Pharmacia Biotech, Piscataway, NJ, USA</td><td></td><td>Multigel caster</td></tr><tr><td>Voyager DE STR MALDI-TOF MS&amp;nbsp;</td><td>ABI, Foster City,CA</td><td></td><td>MALDI-MS PMF</td></tr><tr><td>MALDI-TOF-TOF</td><td>Autoflex III, Bruker</td><td></td><td>MALDI-MS/MS mass spectrometer</td></tr><tr><td>LTQ-OrbiTrap Velos Pro ETD</td><td>Thermo Scientific, Waltham, MA, USA</td><td></td><td>ESI-MS/MS mass spectrometer</td></tr><tr><td>EASY-nano LC system&amp;nbsp;</td><td>Proxeon Biosystems, Odense, Denmark</td><td></td><td>High performance liquid chromatography system</td></tr><tr><td>PepMap C18 trap column&amp;nbsp;</td><td>300 &amp;mu;m i.d. &amp;times; 5 mm length; Dionex Corp., Sunnyvale, CA, USA</td><td></td><td></td></tr><tr><td>RP C18 column</td><td>75 &amp;mu;m i.d., 15 cm length; Dionex Corp., Sunnyvale, CA, USA</td><td></td><td></td></tr><tr><td>KimWipe</td><td>Kimvipe&amp;nbsp;</td><td></td><td>Insoluble paper towel</td></tr><tr><td>Watter</td><td>Made by PURELAB flex instrument</td><td></td><td></td></tr><tr><td>Polytron Model P710/35 homogenizer</td><td>Brinkmann Instruments, Westbury, NY</td><td></td><td></td></tr><tr><td>PDQuest</td><td>Bio-Rad,&amp;nbsp; Hercules, CA</td><td></td><td>2D gel image analysis software</td></tr><tr><td>SEQUEST&amp;nbsp;</td><td>Thermo Proteome Discoverer 1.3 (version No. 1.3.0.339)</td><td></td><td></td></tr><tr><td>DataExplore (ver. 4.0.0.0) software</td><td></td><td></td><td>MS spectrum-processing software</td></tr><tr><td>Mascot software</td><td></td><td></td><td>PMF-based protein searching software&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Mascot software</td><td></td><td></td><td>MS/MS-based protein searching software</td></tr><tr><td>Proteome Discoverer software v.1.3 beta</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Xcalibur software v.2.1</td><td></td><td></td><td>MS/MS data-acquired management software&amp;nbsp;</td></tr><tr><td>Uniprot version 201410.1_HUMAN.fasta</td><td></td><td></td><td>Human protein database</td></tr><tr><td>SEQUEST (version No. 1.3.0.339)&amp;nbsp;</td><td></td><td></td><td>MS/MS-based protein searching software I</td></tr><tr><td>MASCOT (version 2.3.02)&amp;nbsp;</td><td></td><td></td><td>MS/MS-based protein searching software II</td></tr><tr><td>C18 ZipTip microcolumn</td><td>Millipore</td><td></td><td></td></tr><tr><td>PeptideMass Standard kit&amp;nbsp;</td><td>Perspective Biosystems</td><td></td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo&amp;nbsp;Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>2-D Quant Kit</td><td>GE Healthcare</td><td>80-6483-56</td><td></td></tr><tr><td>BIS-ACRYLAMIDE</td><td>AMRESCO</td><td>0172</td><td></td></tr><tr><td>ACRYLAMIDE</td><td>AMRESCO</td><td>0341</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Thiourea</td><td>Sigma-Aldrich</td><td>T8656</td><td></td></tr><tr><td>Urea</td><td>VETEC</td><td>V900119</td><td></td></tr><tr><td>SDS</td><td>AMRESCO</td><td>0227</td><td></td></tr><tr><td>CHAPS</td><td>AMRESCO</td><td>0465</td><td></td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>M146</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>AMRESCO</td><td>M133</td><td></td></tr><tr><td>Trypsin</td><td>Promega, Madison, WI, USA</td><td>V5111</td><td></td></tr><tr><td>IPG buffer pH 3-10, NL</td><td>GE Healthcare</td><td>17-6000-87</td><td></td></tr><tr><td>Immobiline Dry Strip pH 3-10NL,18cm</td><td>GE Healthcare</td><td>17-1235-01</td><td></td></tr></tbody></table>

two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome

人垂体腺瘤 (PA) 是一种常见的肿瘤, 发生在人垂体腺在下丘脑-垂体靶器官轴系统, 并可分为临床功能或非功能性 PA (平安险和消防协会)。由于与平安险相比, 在血液中几乎没有升高激素, 因此, 消防协会很难早期诊断和治疗。我们的长期目标是使用蛋白质组学方法发现可靠的生物标志物, 以澄清 PA 分子机制和识别有效的诊断, 预后标志和治疗目标。本文介绍了一种有效的二维凝胶电泳 (2DE) 结合质谱 (MS) 方法对人体 PA 蛋白质组进行分析, 包括制备样品、2D 凝胶电泳、蛋白质可视化、图像分析、凝胶胰蛋白酶消化, 肽质指纹图谱 (PMF) 和串联质谱 (ms/毫秒)。2维凝胶电泳基质辅助激光解吸/电离质谱 PMF (2 MALDI PMF), 2 MALDI ms/毫秒, 2 de 液相色谱 (LC) ms 和 ms 程序已成功地应用于对消防协会蛋白质组的分析。利用高灵敏度质谱仪, 在对复杂 PA 组织的分析中, 在每个2D 凝胶点上都发现了许多蛋白质, 并采用2的 LC-ms/毫秒方法, 以最大限度地提高人类 PA 蛋白质组的覆盖率。

1. 2 MALDI MS PMF:使用上面描述的实验过程, 从 FSH 表达的消防协会组织 (女性; 50 岁, 促肾上腺皮质激素 (-), 生长激素 (-), 泌乳素 (-), LH (-), FSH (+) 和 TSH (-)) 中提取150µg 蛋白, 并排列18厘米IPG 条 (pH 3-10 NL) 和一个大格式的 SDS 页凝胶, 然后可视化银染色。我们获得了一个可重现和良好的2DE 凝胶模式的消防协会组织蛋白质组 (图 1), 平?...

Watch this Scientific Journal Video about Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome at JoVE.com

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

在这里, 我们提出了一个二维凝胶电泳 (2DE) 与质谱 (MS) 分离和鉴定人垂体腺瘤组织蛋白质组, 这是一个良好的和可重复的2DE 模式。在使用高灵敏度 MS 分析复杂的肿瘤蛋白质组时, 每2DE 点都能观察到许多蛋白质。

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

two-dimensional-gel-electrophoresis-coupled-with-mass-spectrometry

Research

JoVE Journal

Cancer Research

13.4K 次观看.  Central South University. 在这里, 我们提出了一个二维凝胶电泳 (2DE) 与质谱 (MS) 分离和鉴定人垂体腺瘤组织蛋白质组, 这是一个良好的和可重复的2DE 模式。在使用高灵敏度 MS 分析复杂的肿瘤蛋白质组时, 每2DE 点都能观察到许多蛋白质。

视频: Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

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

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

免疫与感染

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

蛋白质组学设施提交和后续数据分析的TMT样品制备

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

神经科学

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

生物学

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

使用基于多反应监测的人类脑组织蛋白质的多反应监测工作流程定量蛋白质组学工作流程

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
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Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
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2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
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Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
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Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
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You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
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二维凝胶电泳

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...