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。

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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

13.3K 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

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length.

使用凝胶内杂交来定量端粒长度的修饰末端限制片段分析

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere ...

科研

癌症研究

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

利用成像质谱技术捕获组织与细胞之间的小分子通信

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Three Differential Expression Analysis Methods for RNA Sequencing: limma, EdgeR, DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education.
Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2.
Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.

A detailed protocol of differential expression analysis methods for RNA sequencing was provided: limma, EdgeR, DESeq2.

RNA 测序的三种微分表达分析方法:利马、边缘、DESeq2

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...