作者承认匹兹堡启动经费的大学和美国国立卫生研究院，NIGMS R01补助（GM 117191-01）到RASR。

伦理声明:小鼠从一个独立的，非盈利性的生物医学研究机构购买和放置在实验室动物资源在匹兹堡大学的分部。所有动物方案都得到了机构动物护理和使用委员会在美国匹兹堡大学获得批准。 1.蛋白提取和多肽的产生化学标记<ol><li> 从组织，细胞，或体液中提取的蛋白质。 <ol><li>与使用机械均化器8M尿素（500μL）匀化60-90毫克在磷酸盐缓冲盐水组织（ 例如脑，心脏，和肝）（1×PBS）的。蛋白酶或磷酸酶抑制剂如果需要可加入到缓冲液中。在这个项目中，他们并没有使用。使用下面的参数用于均化器:裂解矩阵A珠，4米/秒，20秒。心脏组织可能需要均质化的多达9个周期。 注:蛋白酶或磷酸酶抑制剂<html> S可以根据需要添加到缓冲器。他们不是在这项研究中使用。 <ol><li>从裂解管去除组织匀浆并转移到微离心管中。用PBS冲洗100-500μL裂解管有8M尿素和结合用组织匀浆的漂洗溶液。 </li></ol></li><li>离心匀浆组织（9447×g下，4℃，15分钟），并收集上清液。 </li></ol></li><li> 确定使用根据制造商的说明二喹啉甲酸（BCA）测定蛋白质浓度。 注意:如果蛋白质浓度太高进一步的稀释用缓冲液是必需的。将得到的浓度从这些组织样品在6-13微克/微升之间。 <ol><li>可选:添加一个内部质量控制标准（ 例如，牛酪蛋白的α或其它外源蛋白质）与比1微克标准:100微克蛋白样品。 </li></ol></li></ol>jove_title"&gt; 2。消解<ol><li>补充蛋白质的100微克（100×10 -6克）从每个样品单独标记的微离心管中。的体积依赖于蛋白质浓度（参见步骤1.2）。 </li><li>添加二硫苏糖醇（DTT）（40:1试剂加入样品摩尔比），各样品中。孵育在37℃下2小时。对摩尔比计算是基于离牛血清白蛋白的蛋白质质量（BSA），其为66.5×10 3克/摩尔。 </li><li>添加碘乙酰胺（IAM）（80:1试剂加入样品摩尔比），各样品中。在冰上在黑暗中2小时。该反应进行2个小时，以防止副反应在冰上进行。 </li><li>添加L-半胱氨酸（40:1试剂加入样品摩尔比），各样品中。在室温下孵育30分钟。 </li><li>添加20mM Tris缓冲用10mM的CaCl 2（pH为8.2）稀释到尿素2 M的最终浓度</li><li>添加L-1-甲苯磺酰-2-苯乙基氯甲基酮（TPCK）处理的胰蛋白酶（50:1个基板与酶的摩尔比），以每个样品，并在37℃下培育24个小时。 </li><li>通过快速冷冻在液氮中并储存样品在-80℃直到进一步处理淬灭蛋白消化。 </li></ol> 3.样品脱盐<ol><li>通过添加甲酸（FA）重新酸化样品。加入足够的FA，以获得0.1％的终浓度。如果样本最初在-80°C，在冰上解冻样品重新酸化前。 </li><li>去盐使用平衡-C 18亲水亲油（HLB）10毫克墨盒如先前所述肽描述16。 </li><li>干燥通过真空离心（5乇，45℃，77×g）离心所述样本以〜10μL的体积。 </li></ol> 4.二甲基化标记（N-末端） <ol><li>重建在1％乙酸的肽（0.25微克/微升）溶解在高效液相色谱法（HPLC）或纳米纯级水。删除50μ克部分到一个新的微离心管（1.5毫升），并存储在-80℃下的余数。 </li><li>添加8 60毫米（4％）的CHμL2 O（37％重量/体积）或60毫米（4％）13 CD 2 O（20％重量/体积）分别向样品用于标记与轻或重二甲基化， 。通常情况下，试剂的4微升每25微克肽（步骤4.2，4.3，4.6）加入。 </li><li>加入8微升的24mM的NaBH 3 CN或24 mM的NaBD 3 CN到标记有分别轻或重二甲基化，将样品。 </li><li>涡旋并摇动在管式摇床在室温下〜10分钟。 </li><li>通过5分钟，然后加入1％氨水（〜28-30％V / V）的16μL淬灭反应。通常情况下，试剂的8微升每肽的25微克加入。 </li><li>再酸化通过添加8微升5％FA的反应混合物（98％V / V）。 </li><li>合并的轻和重多肽二甲基化（ 图1），以生成总的六个样品。 见表1 </strong&gt;了解用于该研究的样品标记和池的描述。 </li><li>根据步骤3.2和3.3进行样品脱盐。 </li></ol> 5.等压标记（赖氨酸残基） <ol><li>在100mM三乙基碳酸氢铵（TEAB）缓冲液（pH〜8.5）中重构100微克二甲基化的肽（1微克/微升）。 </li><li>准备在制造商的方案所述同量异序的试剂（参见材料/试剂的表 ）。 </li><li>添加溶解的同量异序的试剂（41μL）的肽，涡旋〜10秒。摇上的微离心管摇床〜1个小时的样品。猝灭8μL羟胺（10％W / V）并孵育样品在室温下15分钟的反应。 </li><li>池6个标记的样品到直至进一步使用单一混合物和脱盐（步骤3.1-3.3）或存储在-80℃。 注:为提高蛋白质组覆盖面和深度，多维分离策略建议如强阳离子交换（SCX）。 </li></ol> 6.强阳离子交换<ol><li>执行SCX按照制造商的方案（见材料表 ）。 <ol><li>制备〜1毫升甲酸铵溶液中（20mM，40毫米，60毫米，80毫米，100毫米，150毫米，250毫米，与500毫摩尔）用10％乙腈（ACN），0.1％FA（pH为3）。 </li><li>除去从移液管尖的顶部的红色盖和填料浆料，以确保包装材料是向移液管尖端的底部挖掘移液管尖端。 注:关键:不要扔掉吸管尖端，直到协议结束。 </li><li>将离心适配器到微离心管中（2mL）中的顶部和固定内的移液管尖端。 </li><li>添加SCX重组缓冲液的150微升至移液器吸头。 </li><li>离心机在室温（4000×g）离心6分钟的移液管尖端。重复此步骤两次，丢弃垃圾。 </li><li>溶解在peptides在SCX重建缓冲液的150μL。确保pH为3。 </li><li>添加肽移液器吸头，离心机（见6.1.5），并保持洗脱液。 </li><li>所述过滤器和吸管转移到一个新的微离心管中（2mL）中，并添加20mM的甲酸铵溶液150μL。离心机在室温（4000×g）离心6分钟，并保持洗脱液。 </li><li>重复步骤6.1.8额外七次，使用甲酸铵的连续浓度（ 即 40毫米，60毫米，80毫米，100毫米，150毫米，250毫米和500毫米）。 </li></ol></li><li>传送八个SCX级分至微量离心管（1.5毫升）中，并通过使用离心蒸发浓缩它们（参见步骤3.3）。 </li></ol> 7.液相色谱-串联质谱法（LC-MS / MS）和MS 3 <ol><li>重构质谱级水中肽用0.1％FA，过滤，并放入自动取样管形瓶中。筛选肽FOLLOWS: <ol><li>重构的肽添加到含有0.65微米的过滤器的微离心管中（见材料表 ）。 </li><li>以12,000×g离心1分钟离心肽。删除过滤器，丢弃，并添加过滤肽进入自动取样瓶中。 </li></ol></li><li>用0.1％FA（B）3％，含0.1％FA（A）（V / V）和ACN 100％ACN:制备流动相的缓冲液如下。 </li><li>注入每一SCX级分样品的6μL到填充到2厘米与C 18的材料（5微米，200埃孔径大小）捕获柱。 注:陷阱样品清洗如下:3分钟，0％B;使用2D液相色谱系统3微升/分钟（见材料表 ）。 </li><li>运行的分析分离方法。使用填充有C 18的材料75微米内径x13.2厘米激光拉尖熔融石英毛细管柱（5微米，100埃）。梯度是:0-5分钟，10％B; 5-40分钟，10-15％B; 40-90分钟，15-25％B; 90-115分钟，25-30％B; 115-130分钟，30-60％B; 130-135分钟，60-80％B; 135-145分钟，80％B，145-150分钟，80-10％B; 150-180分钟，10％B; 300标准升/分钟，180分钟。 </li><li>分析分离法运行期间运行质谱仪进行数据采集。 注意:是用于MS调查扫描的参数如下:400-1,600 M / Z，60000分辨率，自动增益控制（AGC）目标1×10 6个离子，最大喷射时间500毫秒。对于CID-MS / MS参数如下:顶1-7或8-14顶取决于数据采集（DDA），3 米/ z分离宽度，500最小信号，35％归一化的碰撞能量（NCE），0.25激活q ，10毫秒的激活时间，3×10 4 AGC，50毫秒最大喷射时间。对于HCD-MS 3参数如下:200最小信号，4 米/ z分离宽度，60％NCE，0.1激活时间，3×10 5 AGC，250毫秒IT，300-1,300 M / Z MS / MS的选择范围，排除的未分片的母离子。 </li><lI&gt;两者的顶部1-7和8-14顶部运行执行一式两份每个分析。 注:样品在含有轨道阱或tribrid质谱仪的仪器的一个较新的模型上运行，DDA参数会变化，并且可以被优化，以包括更多的MS / MS和MS 3次扫描。另外，对于tribrid仪器，同步前体选择（SPS）-MS 3应当被采用。 </li></ol> 8.数据分析16 <ol><li>搜索用蛋白质分析软件对合适的数据库，如:鼠标如Uniprot的RAW文件。 注:这是为了寻找轻型和重型二甲基化肽创建两个工作流程的每个RAW文件非常重要。 </li><li>搜索使用以下参数的RAW文件:具有两个错过裂解，肽质量范围300-6,000道尔顿，15ppm的母体质量公差，1沓破碎耐受胰蛋白酶。静态变形:二甲基化光（28.031，肽N-末端）或heavy二甲基化（36.076道尔顿，肽的N-末端），脲基（57.021沓，C）。 注:有时重峰二甲基化是〜7沓（35.069道尔顿，肽N-末端）是从光二甲基化峰值并且因此应被纳入重二甲基化的肽的搜索工作流程。动态修改:氧化（15.995道尔顿，M），6-PLEX同量异序标签（229.163道尔顿，K）。包括诱饵数据库搜索产生错误发现率（ 例如 1和5％）和定量节点搜索同量异位标记的报告物离子强度。 </li><li>统计<ol><li>导出的数据到电子表格程序，以归一化蛋白报告离子的值。对于每个蛋白，由内标物的内标或原始报告物离子强度的中值比率划分任一中值报告物离子的比例或原始报告物离子强度，分别。使用适当的统计软件，例如电子表格程序，Pers的EUS，或R，以确定样品条件中显著变化。 </li></ol></li></ol>

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<li>Everley, R. A., Kunz, R. C., McAllister, F. E., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+Throughput+in+Targeted+Proteomics+Assays%3A+54-Plex+Quantitation+in+a+Single+Mass+Spectrometry+Run%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Increasing Throughput in Targeted Proteomics Assays: 54-Plex Quantitation in a Single Mass Spectrometry Run.</a> Anal. Chem. 85 (11), 5340-5346 (2013).</li>
<li>Gu, L., Robinson, R. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+endogenous+measurement+of+S-nitrosylation+in+Alzheimer%27s+disease+using+oxidized+cysteine-selective+cPILOT%5BTitle%5D)+AND+%22Analyst%22%5BJournal%5D)">High-throughput endogenous measurement of S-nitrosylation in Alzheimer's disease using oxidized cysteine-selective cPILOT.</a> Analyst. 141 (12), 3904-3915 (2016).</li>
<li>Cao, Z., Evans, A. R., Robinson, R. A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MS3-based+quantitative+proteomics+using+pulsed-Q+dissociation%5BTitle%5D)+AND+%22Rapid+Commun+Mass+Spectrom%22%5BJournal%5D)">MS3-based quantitative proteomics using pulsed-Q dissociation.</a> Rapid Commun Mass Spectrom. 29 (11), 1025-1030 (2015).</li>
<li>Swaney, D. L., Wenger, C. D., Coon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Value+of+Using+Multiple+Proteases+for+Large-Scale+Mass+Spectrometry-Based+Proteomics%5BTitle%5D)+AND+%22J.+Proteome+Res%22%5BJournal%5D)">Value of Using Multiple Proteases for Large-Scale Mass Spectrometry-Based Proteomics.</a> J. Proteome Res. 9 (3), 1323-1329 (2010).</li>
<li>McAlister, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MultiNotch+MS3+Enables+Accurate%2C+Sensitive%2C+and+Multiplexed+Detection+of+Differential+Expression+across+Cancer+Cell+Line+Proteomes%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes.</a> Anal. Chem. 86 (14), 7150-7158 (2014).</li>
</ol>

作者有没有利益争夺。

cPILOT允许多于12个独特的样品的同时测量。为了确保在肽的两个N-末端和赖氨酸残基的成功标记，就必须有正确的pH为每个组反应和首先执行二甲基化反应的肽标记。在N-末端选择性二甲基化是通过在〜2.5的pH（±0.2）中进行。这是通过利用氨基基团的pKa的对赖氨酸和差的N-末端来实现。在pH 2.5，赖氨酸是不活动的（PKA〜10.5）;然而，如果pH在温和的酸性（ 即 pH为5-7）或碱性的，这两个N端和赖?...

蛋白质组学通常涉及用于更好地了解疾病过程许多样品，酶动力学，翻译后修饰，响应于环境刺激，响应于治疗性治疗，生物标志物发现，或药物机制的分析。可以使用定量方法测量通过样品中的蛋白质水平的相对差异，并且可以是无标记的，或涉及同位素标记（代谢，化学或酶）。稳定同位素标记方法已越来越流行，因为它们允许多个样品同时分析，并且适用于从不同的细胞，组织，体液，或整个生物体的样本。同位素标记方法1，2，3，4，5，6，7增加实验的吞吐量，同时减少了采集时间，成本和实验误差。这些方法使用前体的质谱测量从肽峰的蛋白质的相对丰度。相反，同量异序标记试剂8，9，10生成在MS / MS或MS检测任一报告物离子3 11光谱和这些峰被用于对蛋白的相对丰度的报告。 当前状态的最先进的蛋白质组学复用或者是一个10-PLEX 12或12-PLEX同量异位的标签的分析13。增强样品复用（ 即，&gt; 10个样品）的方法已被开发出由我们的实验室组织14，15，16，17，和由他人细胞18的分析</SUP&gt;，19，20，组织21，或靶向肽22。我们开发了所谓的组合的前体同位素标记与同量异序标记（cPILOT）增强的多路复用技术。全球cPILOT是用于获取有关在不同的样品条件（≥12）14的所有蛋白质的相对浓度信息是有用的。 图1示出了一般cPILOT工作流程。胰蛋白酶或Lys-C肽在使用低pH值2的N末端与二甲基化，并在使用高pH 6-PLEX试剂赖氨酸残基选择性地标记。这种策略双打可以与等压试剂，这有助于降低成本实验，另外分析样品的数量，减少实验步骤和时间。 cPILOT是灵活的，我们已经开发了其他的方法来研究氧化翻译后作案fications，包括3-硝基酪氨酸修饰的蛋白质14和与S-亚硝基化（oxcyscPILOT）23含半胱氨酸的肽。我们还开发了一种氨基酸选择性的方法，半胱氨酸cPILOT（cyscPILOT）17。 MS 3获取具有顶离子11或选择性-Y 1 -离子方法15可以帮助减少报告物离子干扰，提高cPILOT的定量精度。在采集方法中使用的MS 3的要求与轨道阱质量分析仪高分辨率仪器虽然低分辨率离子阱仪器也可工作24。 此前，cPILOT已被用于从阿尔茨海默氏症小鼠模型研究肝蛋白16。在这里，我们将介绍如何使用大脑，心脏和肝匀浆研究periphe的角色来执行全球cPILOT分析RY阿尔茨海默病。本实验采用了生物复制。由于cPILOT的多功能性，有兴趣的用户可以使用该技术来研究其他组织为一系列的生物学问题和系统。

<table><tbody><tr><td>Water - MS Grade</td><td>Fisher Scientific</td><td>W6-4</td><td>4 L quantity is not necessary</td></tr><tr><td>Acetonitrile - MS Grade</td><td>Fisher Scientific</td><td>A955-4</td><td>4 L quantity is not necessary</td></tr><tr><td>Acetic Acid</td><td>J.T. Baker</td><td>9508-01</td><td></td></tr><tr><td>Ammonium hydroxide solution (28 - 30%)</td><td>Sigma Aldrich</td><td>320145-500ML</td><td></td></tr><tr><td>Ammonium formate</td><td>Acros Organics</td><td>208-753-9</td><td></td></tr><tr><td>Formic Acid</td><td>Fluka Analytical</td><td>94318-250ML-F</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Pierce Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>Urea</td><td>Biorad</td><td>161-0731</td><td></td></tr><tr><td>Tris</td><td>Biorad</td><td>161-0716</td><td></td></tr><tr><td>Dithiothreiotol (DTT)</td><td>Fisher Scientific</td><td>BP172-5</td><td></td></tr><tr><td>Iodoacetamide (IAM)</td><td>Acros Organics</td><td>144-48-9</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Aldrich, Chemistry</td><td>168149-25G</td><td></td></tr><tr><td>L-1-tosylamido-2 phenylethyl cholormethyl ketone (TPCK)-treated Trypsin from bovine pancreas</td><td>Sigma Aldrich, Life Science</td><td>T1426-100MG</td><td></td></tr><tr><td>Formaldehyde (CH&lt;sub&gt;2&lt;/sub&gt;O) solution; 36.5 - 38% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich, Life Science</td><td>F8775-25ML</td><td></td></tr><tr><td>Formaldehyde (&lt;sup&gt;13&lt;/sup&gt;CD&lt;sub&gt;2&lt;/sub&gt;O) solution; 20 wt % in D&lt;sub&gt;2&lt;/sub&gt;O, 98 atom % D, 99 atom % &lt;sup&gt;13&lt;/sup&gt;C</td><td>Sigma Aldrich, Chemistry</td><td>596388-1G</td><td></td></tr><tr><td>Sodium Cyanoborohydride; reagent grade, 95%</td><td>Sigma Aldrich</td><td>156159-10G</td><td></td></tr><tr><td>Sodium Cyanoborodeuteride; 96 atom % D, 98% CP</td><td>Sigma Aldrich, Chemistry</td><td>190020-1G</td><td></td></tr><tr><td>Strong Cation Exchange (SCX) spin tips sample prep kit</td><td>Protea BioSciences</td><td>SP-155-24kit</td><td></td></tr><tr><td>Triethyl ammonium bicarbonate (TEAB) buffer</td><td>Sigma Aldrich, Life Science</td><td>T7408-100ML</td><td></td></tr><tr><td>Isobaric Tagging Kit (TMT 6 plex) - 6 reactions (1 x 0.8 mg)</td><td>Thermo Fisher Scientific</td><td>90061</td><td></td></tr><tr><td>Hydroxylamine hydrochloride</td><td>Sigma Aldrich, Chemistry</td><td>255580-100G</td><td></td></tr><tr><td>Standard vortex mixer</td><td>Fisher Scientific</td><td>2215365</td><td>any mixer can be used</td></tr><tr><td>Oasis HLB 1 cc (10 mg) extraction cartridges</td><td>Waters</td><td>186000383</td><td>These are C&lt;sub&gt;18&lt;/sub&gt; cartridges</td></tr><tr><td>Visiprep SPE vacuum manifold, DL (disposable liner), 24 port model</td><td>Sigma Aldrich</td><td>57265</td><td>A 12 port model is also sufficient</td></tr><tr><td>Speed-vac</td><td>Thermo Scientific</td><td>SPD1010</td><td>any brand of speed vac is sufficient</td></tr><tr><td>Water bath chamber</td><td>Thermo Scientific</td><td>2825/2826</td><td>Any brand of&amp;nbsp; a water bath chamber with controlled temperatures is sufficient.</td></tr><tr><td>Mechanical Homogenizer (&lt;em&gt;i.e.&lt;/em&gt; FastPrep-24 5G)</td><td>MP Biomedicals</td><td>116005500</td><td></td></tr><tr><td>Eksigent Nano LC - Ultra 2D with Nano LC AS-2 autosampler</td><td>Sciex</td><td>-</td><td>This model is no longer available. Any nano LC with an autosampler is sufficient.</td></tr><tr><td>LTQ Orbitrap Velos Mass Spectrometer</td><td>Thermo Scientific</td><td>-</td><td>This model is no longer available. Other high resolution instruments (&lt;em&gt;e.g.&lt;/em&gt; Orbitrap Elite, Orbitrap Fusion, or Orbitrap Fusion Lumos) can be used.</td></tr><tr><td>Protein software (&lt;em&gt;e.g.&lt;/em&gt; Proteome Discoverer)</td><td>Thermo Scientific</td><td>IQLAAEGABSFAKJMAUH&amp;nbsp;</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>AL54</td><td></td></tr><tr><td>Stir plate</td><td>VWR</td><td>12365-382</td><td>Any brand of stir plates are sufficient</td></tr><tr><td>pH meter (Tris compatiable)</td><td>Fisher Scientific (Accumet)</td><td>13-620-183</td><td>Any brand of a pH meter is sufficient</td></tr><tr><td>pH 10 buffer</td><td>Fisher Scientific</td><td>06-664-261</td><td>Any brand of pH buffer 10 is sufficient</td></tr><tr><td>pH 7 buffer</td><td>Fisher Scientific</td><td>06-664-260</td><td>Any brand pH buffer 7 is sufficient</td></tr><tr><td>1.5 mL eppendorf tubes, 500 pk</td><td>Fisher Scientific</td><td>05-408-129</td><td>Any brand of 1.5 mL eppendorf tubes are sufficient</td></tr><tr><td>0.6 mL eppendorf tubes, 500 pk</td><td>Fisher Scientific</td><td>04-408-120</td><td>Any brand of 0.6 mL eppendorf tubes are sufficient</td></tr><tr><td>0.65 &amp;micro;m Ultrafree MC DV centrifugal filter units</td><td>EMD Millipore</td><td>UFC30DV00</td><td></td></tr><tr><td>2 mL microcentrifuge tubes, 72 units</td><td>Thermo Scientific</td><td>69720</td><td></td></tr><tr><td>C&lt;sub&gt;18&lt;/sub&gt; packing material (5&amp;nbsp;&amp;micro;m, 100 &amp;Aring;)</td><td>Bruker</td><td>PM5/61100/000</td><td>This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient</td></tr><tr><td>C&lt;sub&gt;18&lt;/sub&gt; packing material (5&amp;nbsp;&amp;micro;m, 200 &amp;Aring;)</td><td>Bruker</td><td>PM5/61200/000</td><td>This item is no longer available from Bruker. Alternative packing material with listed specifications will be sufficient</td></tr></tbody></table>

enhanced sample multiplexing of tissues using combined precursor isotopic labeling and isobaric tagging (cpilot)

有日益增加的需求，分析了大量生物标本用于疾病的理解和生物标志物发现。定量蛋白质组学策略，允许多个样品的同时测量已经普及，大大降低实验成本和时间。我们实验室开发了一种称为合并的前体同位素标记和同量异位素标记（cPILOT）技术，这增强了传统的同位素标记或同量异位的标记方法中的样品的复用。全球cPILOT可以应用于从细胞，组织，体液，或整个生物体始发样品和给出了关于在不同的样品条件相对蛋白质丰度的信息。 cPILOT使用低pH值的缓冲液条件的工作方式是1），以选择性地dimethylate肽的N-末端，并使用高pH缓冲液条件2）标记的赖氨酸残基的伯胺与市售的同量异序的试剂（参见材料/试剂的表 ）。度可用的样本复用取决于所使用的前体的标签的数量和同量异序标记试剂。这里，我们提出使用轻和重的二甲基六-PLEX等压试剂组合以分析来自小鼠组织的12个样品在一次分析中一个12-PLEX分析。增强的复用是用于减少实验时间和成本，并且更重要的是，允许在许多样品条件（生物学重复，疾病阶段，药物治疗，基因型，或纵向的时间点）相比具有较少实验偏差和误差有帮助的。在这项工作中，全球cPILOT方法来分析大脑，心脏和跨越生物学重复肝组织从阿尔茨海默氏症小鼠模型和野生型对照。全球cPILOT可应用于研究其他生物过程和适于增加样本复用大于20个样本。

cPILOT使用基于胺的化学化学键在N-末端和赖氨酸残基的肽标签和增强样品复用能力。 图2示出了从从阿尔茨海默氏病小鼠模型中和野生型对照脑，心脏和肝组织中的12-plex的cPILOT分析所获得的代表性MS数据。如表1所示，对于阿尔茨海默氏病和野生型小鼠的两个生物学重复被包括在这12个复合体的分析。 图2A示出了一个双电荷峰值对由指?...

Watch this Scientific Journal Video about Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT at JoVE.com

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

合并前体同位素标记和等压标记（cPILOT）是定量蛋白质组学战略，增强同量异位标记的样品复用能力。这个协议描述cPILOT的对组织从阿尔茨海默氏病小鼠模型中和野生型对照的应用。

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

enhanced-sample-multiplexing-tissues-using-combined-precursor

Research

JoVE Journal

Biochemistry

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

6.9K 次观看.  University of Pittsburgh. 合并前体同位素标记和等压标记（cPILOT）是定量蛋白质组学战略，增强同量异位标记的样品复用能力。这个协议描述cPILOT的对组织从阿尔茨海默氏病小鼠模型中和野生型对照的应用。 

视频: Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

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) ...

科研

生物化学

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 ...

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 ...

癌症研究

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

化学

Highly-Multiplexed Tissue Imaging with Raman Dyes

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

使用拉曼染料进行高度多重组织成像

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. ...

生物工程

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

免疫与感染

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

生物学

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

量化和站点本地化两个翻译后修改的同步亲和力丰富

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

摘要

探索更多视频

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

蛋白质组学设施提交和后续数据分析的TMT样品制备

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

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用

使用拉曼染料进行高度多重组织成像

冷冻组织部分多光谱荧光成像的快速方法

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

量化和站点本地化两个翻译后修改的同步亲和力丰富

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

摘要

探索更多视频

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

蛋白质组学设施提交和后续数据分析的TMT样品制备

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

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

树脂辅助捕获与同量异位串联质量标签标记相结合，用于蛋白质硫醇氧化的多重定量

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

使用拉曼染料进行高度多重组织成像

冷冻组织部分多光谱荧光成像的快速方法

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

量化和站点本地化两个翻译后修改的同步亲和力丰富

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用