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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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Multiple+Reporter+Ions+from+a+Single+Isobaric+Reagent+Increases+Multiplexing+Capacity+for+Quantitative+Proteomics.">Generation of Multiple Reporter Ions from a Single Isobaric Reagent Increases Multiplexing Capacity for Quantitative Proteomics.</a> Analytical Chemistry. 87 (19), 9855-9863 (2015).</li><li>Everley, R. A., Kunz, R. C., McAllister, F. E., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+Throughput+in+Targeted+Proteomics+Assays:+54-Plex+Quantitation+in+a+Single+Mass+Spectrometry+Run.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+endogenous+measurement+of+S-nitrosylation+in+Alzheimer's+disease+using+oxidized+cysteine-selective+cPILOT.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS3-based+quantitative+proteomics+using+pulsed-Q+dissociation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+Using+Multiple+Proteases+for+Large-Scale+Mass+Spectrometry-Based+Proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiNotch+MS3+Enables+Accurate,+Sensitive,+and+Multiplexed+Detection+of+Differential+Expression+across+Cancer+Cell+Line+Proteomes.">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端和赖氨酸残基将被二甲基化。此外，如果是在低pH值首先进行同量异位标记，N-末端将具有同量异序标签和赖氨酸残基将被二甲基化。这可以导致更少的片段被选择用于MS 3为b-离子将需要选择。二甲基的相对成本相比商业等压试剂通货膨胀的反应是价格便宜。尽管人们可以每100微克使用整个等压试剂瓶，我们用每100微克的试剂瓶的一半，可比的标记效率也有成功。这有助于减少显著为个人cPILOT实验的成本，并允许分析更多的样品。还需要注意的是其他的等压标记试剂可以发生在这个协议中使用的等压试剂可以作为我们先前已经证实14是很重要的。为确保高标记和标记效率，以试剂快速添加到样品是很重要的。这将允许样品具有大致相同的反应时间。对于定量的目的，每个样品应被视为相同尤其之前在二甲基化和同量异序标记步骤合并样本。最后，应该指出的是，在requir的初始步骤同时与许多样品的工作ES细心的用户技巧和注意样品处理。 最理想的情况是，以获得报告物离子在整个12个样品的每一个混合物蛋白。然而，这不是大量蛋白质的情况下。有用于cPILOT定量信息和其它增强复用接近检测肽的数量取决于多种因素，包括样品类型，样品分级和处理步骤，MS数据采集方法和仪器类型。虽然这两个二甲基化和同量异序标记的步骤具有〜95-99％的高肽标记效率，仍然〜这将不包含报告物离子信息的MS 3数据的20％。这是部分由于用胰蛋白酶，其产生导致没有掺入同量异位的试剂精氨酸 - 封端的肽。这可以使用其它酶，如型lysC来解决，在蛋白质鉴定25一个潜在的折衷。还重二甲基化的肽，选择用于碎片峰可以是M或M-1峰，其具有不同的强度，并会影响在MS 3阶段观察到的报告物离子强度。因此，有可能是没有对于一些样品检测到某些低强度的报告物离子。这种情况下经常被用于通常被隔离MS 3个步骤的低强度的MS / MS片段观察到。对这个问题的理想解决方案已被纳入tribrid质谱仪，其中，代替使用单级选择的MS 3时 ，使用多凹口或多个MS / MS碎片26。 有很多在cPILOT方法的多功能性尤其是关于可在一次分析中进行比较（ 即 ，最多20个与商业同量异序的试剂）的样本的数目和所使用的类型的组织。我们表明，它是很容易获得的脑，心脏准确的定量信息，并肝组织中的疾病的情况下进行同样的分析。这种方法，类似于其它复用方法，允许多个样品的分析一次，并且适用于从细胞，组织，体液，或整个生物体始发样品。此外，cPILOT标记的肽都能够使用一个低24或高分辨率（60,000）仪器进行分析。相比较而言，获得使用其他的复用方法的成功测量可以被限制为特定类型的样品（ 即细胞）18，可能需要具有非常高的分辨率20，或获得稳定同位素标记的工具。 cPILOT是伟大的兴趣疾病的认识，生物标志物发现，应对药物或治疗干预，或在多个时间点的纵向变化的用户。此外，对于大型鸟枪蛋白质组学临床样本（其中N是大的，几百到几千），CP的分析ILOT也可能是合适的，以帮助减少实验成本和时间。在未来，cPILOT将扩大通过使用现有的和新的同位素和同量异序标记物复采样的更大的数字。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (CH2O) solution; 36.5 - 38% in H2O</td>
			<td>Sigma Aldrich, Life Science</td>
			<td>F8775-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (13CD2O) solution; 20 wt % in D2O, 98 atom % D, 99 atom % 13 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)&#160;</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 1cc (10 mg) &#160;&#160;extraction cartridges</td>
			<td>Waters</td>
			<td>186000383</td>
			<td>These are C18 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&#160; a water bath chamber with controlled temperatures is sufficient.</td>
		</tr>
		<tr>
			<td>Mechanical Homogenizer (i.e. 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 (e.g. Orbitrap Elite, Orbitrap Fusion, or Orbitrap Fusion Lumos) can be used.</td>
		</tr>
		<tr>
			<td>Protein software (e.g. Proteome Discoverer)</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGABSFAKJMAUH&#160;</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>&#160;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&#160; is sufficient</td>
		</tr>
		<tr>
			<td>1.5 mL eppendorf tubes, 500pk</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, 500pk</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&#181;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>C18 packing material (5 &#181;m, 100 &#197;)</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>C18 packing material (5 &#181;m, 200 &#197;)</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示单个二甲基被并入肽4 M / Z间距隔开。两者的光并且在该对重二甲基化的峰是独立分离和零散与CID。对于每个二甲基化的肽的MS / MS数据显示在图2B和C。搜索结果表明，该PAI峰的R的包含T（二甲基）ELNYFAK（同量异位的标签6）蛋白磷酸甘油酸激酶1，最激烈的碎片离子是Y 3+是用于既轻和重峰二甲基化的类似肽。这些峰对HCD-MS 3进一步分离和报告物离子（M / Z 126-131）被观察为示于图2D和2E。 MS 3谱的两组是必要的，以获取有关12个样品的信息。在这个例子中，记者离子比率（AD / WT）（阿尔茨海默氏病/野生型对照）的脑，肝脏，和心脏组织是跨两个生物学重复类似。每次比较的倍数变化值表明，在脑和心脏磷酸甘油酸激酶1个水平在AD小鼠的要求较高，而在肝中的水平更低。 <img alt="图1" src="/files/ftp_upload/55406/55406fig1.jpg" /> Figu重1:蛋白质组学工作流程中使用cPILOT。作为一个例子，这个工作流程列出的12周单独的样品进行分析。从组织，细胞，或体液的蛋白提取和合适的蛋白质标准（ 例如牛的α-酪蛋白）中的溶液。蛋白质使用胰蛋白酶消化。肽在N-末端通过使用轻或重的二甲基（pH约2.5）和在赖氨酸残基通过使用TMT 6 -plex（pH为〜8.5）标记的。标记的肽汇集成一个单一的混合物中，并受到SCX RP-LC-MS / MS和MS 3。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55406/55406fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55406/55406fig2.jpg" /> 图2:从脑，心脏，和阿尔茨海默氏病小鼠模型中和野生型对照的肝组织的肽的实施例cPILOT数据。 </s仲&gt;前体数据（A）示出了轻链和重二甲基化肽，通过在m / z 643.854和647.875峰表示。选择这些肽，分离和分散，从而生成CID-MS / MS谱（B和C），其提供肽鉴定。一个额外的选择，分离，并在MS / MS阶段中的轻和重多肽二甲基化的最强烈的碎片离子的碎裂产生的HCD-MS谱3（D和E），分别。肽序列是T（二甲基）ELNYFAK（同量异位的标签6）和属于磷酸甘油酸激酶1 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55406/55406fig2large.jpg" target="_blank">请点击此处以查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="6">等压试剂0; </td></tr><tr><td></td><td> 126 </td><td> 127 </td><td> 128 </td><td> 129 </td><td> 130 </td><td> 131 </td></tr><tr><td>光二甲基化</td><td> WT 一 </td><td>广告b </td><td> WT </td><td>广告</td><td> WT </td><td>广告</td></tr><tr><td></td><td colspan="2">脑</td><td colspan="2">心</td><td colspan="2">肝</td></tr><tr><td>重型二甲基化</td><td> WT </td><td>广告</td><td> WT </td><td>广告</td><td> WT </td><td>广告</td></tr><tr><td></td><td colspan="2">心</td><td colspan="2">肝</td><td colspan="2">脑</td></tr><tr><td colspan="7">所述组织是无论是从一个野生型对照（WT） 一个或阿尔茨海默氏病小鼠（AD） 湾 </td></tr></tbody></table> 表1:AD和WT脑，心脏，和肝组织的cPILOT分组。

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

6.8K 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

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

科研

生物化学

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

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

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

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

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

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

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 ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

使用支撑浮块在透射电子显微镜中制备样品支撑膜

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...