We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

注:此方法之前描述12。 1，蛋白质分离制备1毫克细胞蛋白通过裂解细胞，优选通过物理方法如法国压机，珠击，或超声处理。避免溶菌酶介导的细胞溶解，因为这种酶会混淆质谱测量。 2，三氯乙酸沉淀蛋白加入1倍体积的三氯乙酸（TCA），以4倍体积的冰蛋白和冷却10分钟以沉淀蛋白质。 12,000×g离心5分钟，在4℃下，去除上清。在4℃下重悬沉淀中加入1ml冰冷的丙酮和12,000×g离心5分钟去除上清，颠倒离心管在板凳上，以干燥沉淀15分钟。店铺蛋白小球于-80℃。 3，使蛋白质变性和还原二硫键回复暂停蛋白质〜2 mg / ml的500微升变性和还原缓冲液（或者4M尿素或3％SDS的50mM HEPES pH值为8.5，5mM DTT的）。任选地，包括在缓冲液中的蛋白酶抑制剂。孵育蛋白30分钟，在56℃，然后在室温下10分钟。 4。Aklylate游离巯基，以不可逆扰乱二硫键形成水准备新鲜0.3M的碘乙酰胺。注意！碘乙酰胺是剧毒。加入25微升0.3M的碘乙酰胺（15mM的终浓度），以500μl的蛋白孵育20分钟在黑暗中在室温下进行。通过加入10μl的300mM的DTT（5mM的终浓度DTT）淬灭碘乙酰胺。存储烷基化的蛋白质于-80℃。 5，蛋白质的消化 TCA沉淀的蛋白质（如步骤2中所述），并重新悬浮于1ml的50mM HEPES（pH值8.2），1M尿素。准备赖氨酰endoprotein的原液酶（赖氨酸-C）在水中的2微克/微升的浓度，并添加5微升的蛋白质溶液。为16小时，在室温下孵育混合物。确保最终的赖氨酸-C浓度为10纳克/微升和蛋白质对lysC的比率（W / W）为1/50至1/200。重悬20微克测序级胰蛋白酶在40微升50mM的乙酸中，添加5微升（10微克胰蛋白酶）对赖氨酸-C消化物，并孵育6小时，在37℃下使用相同浓度的蛋白酶和蛋白酶与蛋白质的比例为赖氨酸-C作为用于胰蛋白酶。 6，反相肽提取<ol><li>通过加入三氟乙酸（TFA）至0.5％的最终浓度（pH≈2）酸化肽。附加C18柱，以提取歧管。除了使用肽的装载和洗脱尽可能高的流速所有步骤。 </li><li>湿塔用6ml乙腈（ACN）。用6ml 80％ACN，0.1％TFA洗柱，然后平衡用6毫升的0.1％TFA。不要让列步骤之间的干运行。 </li><li>停止真空压力和负载500的肽的微克到柱上以大约1毫升/分钟的流速。一旦肽已结合在柱上，重新启动真空和洗涤用6ml 0.1％TFA，然后用3毫升的柠檬酸缓冲液（0.09M的柠檬酸，0.23，1M的Na 2 HPO 4，pH 5.5）中。 注意:较高的肽量可以被标记，但C18柱结合能力至少应为2倍左右的肽量更高，以避免样品损失。 </li></ol> 7，柱头肽标记通过还原二甲基化（REDI标签） 如氰化氢在贴标过程中释放在低浓度下，化学罩执行此步骤。 <ol><li>准备将12ml"轻"和"重"REDI缓冲区，以甲基化肽的游离胺。光REDI缓冲由0.8％甲醛和0.12 M氰基硼氢化钠携带氢在日EIR天然同位素分布在柠檬酸缓冲液。重REDI缓冲由0.8％氘代甲醛和0.12 M在柠檬酸缓冲液氘代氰基硼氢化钠。 </li><li>孵育含有肽柱中加入10ml或者轻或重的Redi缓冲区以1毫升/分钟流速的含肽柱，并重复以确保完整的标签。洗柱用6毫升含0.1％TFA，然后用1毫升0.5％的乙酸。 </li><li>停止真空并用1毫升40％ACN，0.5％乙酸，然后用1毫升80％ACN，0.5％乙酸使用约0.5ml /分钟的流速洗脱第一个标记的肽。如果需要，测量单个样品的标记效率通过质谱混合重型和轻型样品前（参见"代表业绩」）。拌1点01重链和轻-标记的肽样品，通过质谱法进行定量。 </li></ol> 8，独立的肽混合物用pH值基本反相（BPRP）色谱&lt;/ P&gt; 碱性pH值反相（BPRP）层析，肽混合物分离成多个级分，这是通过LC-MS/MS独立地分析，以增加蛋白质的覆盖范围。 <ol><li>通过施加增加乙腈浓度的梯度在10mM碳酸氢铵（pH8）中的C18-HPLC柱分级分离肽混合物。开始用5％（V / V）乙腈5分钟，加至35％的ACN 60分钟，然后以90％的ACN 1分钟。降低ACN 5％至重新平衡为9分钟的列之前保留在90％ACN 4分钟。收集馏分96等体积在96 - 孔板（A1至H12）。使用UV检测器在220nm处，而肽的洗脱从柱（10-70分钟对这里所描述的条件下）监测分馏。 </li><li>从孔A1，C1，E1和G1（级分A1）结合级分，从井B1，D1，F1和H1（馏分B1）中，从井A2，C2，E2和G2（级分A2），并相应的剩余部分。取出瑟尔t使用真空离心机。从馏分重悬肽A1，B2，A3，B4，A5，B6，A7，B8，A9，B10，A11，B12和在130微升的1M urea/0.5％的TFA和纯化，如步骤9中所述使用StageTips。店级分B1，A2，B3，A4，B5，A6，B7，A8，B9，A10，B11，A12和在-20℃。 </li></ol> 9。净化肽由走走停停萃取（StageTips） 由包装200微升移液器吸头具有两个磁盘C18为1.07毫米的内径（ID）的制备C18-StageTip 7微柱。把舞台提示到Eppendorf管。使用微量离心与130微升甲醇，然后130 UL 80％ACN，0.5％醋酸冲洗技巧。平衡StageTips用130微升0.1％TFA。转肽混合物以StageTips和洗涤用130微升0.1％TFA，然后加入40μl的0.1％TFA，然后加入40μl乙酸0.5％。第一洗脱的肽用20微升40％乙腈，0.5％乙酸，然后用20微升80％乙腈，0.5％乙酸。合并洗脱液一第二干燥，真空过滤。 10。微细的LC-MS/MS <ol><li>溶解肽在1-5微升5％的甲酸，5％乙腈至约1微克/微升的浓度。解决〜1微克的肽上的100微米×20厘米C18-反相HPLC柱，用6-22％乙腈的0.125％甲酸梯度施加在75或100分钟的〜300升/分钟的流速。 </li><li>通过使用LTQ的Orbitrap Velos的12或类似的液相色谱-质谱联用平台与质谱仪提供高分辨率和高质量精度鉴定的肽。操作质谱仪的数据依赖模式，在轨道阱分析仪获得了完整的MS扫描（分辨率为60,000）。产生线性离子阱MS / MS谱图在全质谱检测到的20种最丰富的离子。对于MS / MS设置自动增益控制（AGC）的目标，以1×10 6的完整的MS和2000。设置最大的离子积累倍至1,000毫秒对于MS和150毫秒的MS / MS分析。从进一步选择从MS / MS进行20-60秒不包括零碎肽前体离子。 </li></ol> 11，MS / MS数据采集通过使用这些参数（ 表1）比较，MS / MS谱图RAW文件到一个理论数据库用算法如SEQUEST 8识别的肽。 表1。肽数据库搜索参数。 <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> 常规参数 </td><td>全胰蛋白酶消化高达2错过分裂</td></tr><tr height="19"><td> 25 ppm的母离子耐受性</td></tr><tr height="19"><td> 1.0大碎片离子耐受性</td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> 静态修改 </sTRONG&gt; </td><td> 57.02146 Da于半胱氨酸，carboxyamidomethylation </td></tr><tr height="19"><td> 28.03130达赖氨酸和肽的N-末端，光二甲基化标签</td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> 动态修改 </td><td> 15.99491达蛋氨酸，氧化</td></tr><tr height="19"><td> 6.03766沓上赖氨酸和肽的N-末端，重二甲基化标签</td></tr></tbody></table><ol><li>滤波器的肽使用的开放阅读框架的一个数据库中的实际和扭转方向1％的错误发现率的方法，如目标诱饵9策略。 </li></ol> 12。肽定量计算重型和轻型双MS1的领域提取离子色谱（MS1峰面积）和肽信噪（S / N）比10。包括肽对只有当他们的平均信号 - 河内SE比上述五。量化的肽的相对丰度的两个样本为相同的肽（MS1峰面积比）的重链和轻版本MS1峰面积的比值在。计算的相对丰度蛋白的中位数MS1峰面积比为在该蛋白质的所有肽。

<ol>
	<li>Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M., Yates III, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+quantitative+proteomic+data+generated+via+multidimensional+protein+identification+technology.">Analysis of quantitative proteomic data generated via multidimensional protein identification technology.</a> Analytical chemistry. 74 (7), 1650-1657 (2002).</li><li>Ong, S. -. E., Blagoev, B., 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=Stable+isotope+labeling+by+amino+acids+in+cell+culture,+SILAC,+as+a+simple+and+accurate+approach+to+expression+proteomics.">Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.</a> Molecular &amp; cellular proteomics: MCP. 1 (5), 376-386 (2002).</li><li>Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nature biotechnology. 17 (10), 994-999 (1999).</li><li>Ross, P. L., 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=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Molecular &amp; cellular proteomics: MCP. 3 (12), 1154-1169 (2004).</li><li>Hsu, J. -. L., Huang, S. -. Y., Chow, N. -. H., Chen, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+dimethyl+labeling+for+quantitative+proteomics.">Stable-isotope dimethyl labeling for quantitative proteomics.</a> Analytical Chemistry. 75 (24), 6843-6852 (2003).</li><li>Chick, J. M., Haynes, P. A., Molloy, M. P., Bjellqvist, B., Baker, M. S., Len, A. C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+rat+liver+membrane+proteome+using+peptide+immobilized+pH+gradient+isoelectric+focusing.">Characterization of the rat liver membrane proteome using peptide immobilized pH gradient isoelectric focusing.</a> Journal of Proteome Research. 7 (3), 1036-1045 (2008).</li><li>Rappsilber, J., Ishihama, Y., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+and+go+extraction+tips+for+matrix-assisted+laser+desorption/ionization,+nanoelectrospray,+and+LC/MS+sample+pretreatment+in+proteomics.">Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.</a> Analytical chemistry. 75 (3), 663-670 (2003).</li><li>Eng, J. K., McCormack, A. L., Yates III, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+correlate+tandem+mass+spectral+data+of+peptides+with+amino+acid+sequences+in+a+protein+database.">An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.</a> Journal of the American Society for Mass Spectrometry. 5 (11), 976-989 (1994).</li><li>Elias, J. 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=Target-decoy+search+strategy+for+increased+confidence+in+large-scale+protein+identifications+by+mass+spectrometry.">Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.</a> Nature methods. 4 (3), 207-214 (2007).</li><li>Bakalarski, C. E., 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=The+impact+of+peptide+abundance+and+dynamic+range+on+stable-isotope-based+quantitative+proteomic+analyses.">The impact of peptide abundance and dynamic range on stable-isotope-based quantitative proteomic analyses.</a> Journal of Proteome Research. 7 (11), 4756-4765 (2008).</li><li>Wilson-Grady, J. T., Haas, W., 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=Quantitative+comparison+of+the+fasted+and+re-fed+mouse+liver+phosphoproteomes+using+lower+pH+reductive+dimethylation.">Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation.</a> Methods (San Diego, Calif.). , (2013).</li><li>Tolonen, A. C., Haas, W., Chilaka, A. C., Aach, J., Gygi, S. P., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+systems+analysis+of+a+cellulosic+biofuel-producing+microbe.">Proteome-wide systems analysis of a cellulosic biofuel-producing microbe.</a> Molecular Systems Biology. 7, 461 (2011).</li><li>Tolonen, A. C., Chilaka, A. C., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+inactivation+in+Clostridium+phytofermentans+shows+that+cellulose+degradation+requires+the+family+9+hydrolase+Cphy3367.">Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367.</a> Molecular Microbiology. 74 (6), 1300-1313 (2009).</li><li>Munoz, J., 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=The+quantitative+proteomes+of+human-induced+pluripotent+stem+cells+and+embryonic+stem+cells.">The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells.</a> Molecular systems biology. 7, 550 (2011).</li><li>Kovanich, D., Cappadona, S., Raijmakers, R., Mohammed, S., Scholten, A., Heck, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+stable+isotope+dimethyl+labeling+in+quantitative+proteomics.">Applications of stable isotope dimethyl labeling in quantitative proteomics.</a> Analytical and bioanalytical chemistry. 404 (4), 991-1009 (2012).</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=Increasing+the+multiplexing+capacity+of+TMTs+using+reporter+ion+isotopologues+with+isobaric+masses.">Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.</a> Analytical chemistry. 84 (17), 7469-7478 (2012).</li><li>Boersema, P. J., Aye, T. T., Van Veen, T. A. B., Heck, A. J. R., Mohammed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triplex+protein+quantification+based+on+stable+isotope+labeling+by+peptide+dimethylation+applied+to+cell+and+tissue+lysates.">Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates.</a> Proteomics. 8 (22), 4624-4632 (2008).</li><li>Wu, Y., 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=Five-plex+isotope+dimethyl+labeling+for+quantitative+proteomics.">Five-plex isotope dimethyl labeling for quantitative proteomics.</a> Chemical communications (Cambridge, England). , (2014).</li><li>Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+peptide+stable+isotope+dimethyl+labeling+for+quantitative+proteomics.">Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.</a> Nature Protocols. 4 (4), 484-494 (2009).</li><li>She, Y. -. M., Rosu-Myles, M., Walrond, L., Cyr, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+protein+isoforms+in+mesenchymal+stem+cells+by+reductive+dimethylation+of+lysines+in+intact+proteins.">Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins.</a> Proteomics. 12 (3), 369-379 (2012).</li><li>Chen, S. -. H., Chen, C. -. R., Chen, S. -. H., Li, D. -. T., Hsu, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+N(&#945;)-acetylated+Peptide+Enrichment+Following+Dimethyl+Labeling+and+SCX.">Improved N(&#945;)-acetylated Peptide Enrichment Following Dimethyl Labeling and SCX.</a> Journal of proteome research. 12 (7), 3277-3287 (2013).</li><li>Sun, Z., 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=Capture+and+Dimethyl+Labeling+of+Glycopeptides+on+Hydrazide+Beads+for+Quantitative+Glycoproteomics+Analysis.">Capture and Dimethyl Labeling of Glycopeptides on Hydrazide Beads for Quantitative Glycoproteomics Analysis.</a> Analytical Chemistry. 84 (20), 8452-8456 (2012).</li></ol>

作者宣称没有竞争的经济利益或其他的利益冲突。

几点让使用还原二甲基化（REDI标签）一个有吸引力的方法，定量蛋白质组学肽的稳定同位素标记:物美价廉标记试剂（试剂成本低于1美元的样品），反应速度快（〜10分钟），无副产物，高再现性（ 图3,4），稳定的反应产物，能够使用任何蛋白酶，和标记的肽的高电离效率。由雷迪化学标记也是有利的相对于代谢标记，因为它不需要株或细胞系具有特定氨基酸营养缺陷型或生长在合?...

测量复杂样品间的许多蛋白质​​的浓度差异是在蛋白质组学面临的核心挑战。越来越多地，这是由标记蛋白在每个样品中不同的同位素标记，结合样品，并用质谱定量浓度差异进行。蛋白质和多肽的稳定同位素标记的方法有几种。15 N标记1和2 SILAC 在体内引入同位素标记的代谢，而ICAT 3的iTRAQ 4，和二甲基化减少5蛋白的提取和消化后添加稳定同位素标记。在这些方法中，还原的二甲基化（雷迪标签）正逐渐成为一种廉价的，可再现的方法来量化在几乎任何类型的样品的蛋白质浓度差异。 雷迪标记包括使肽与甲醛以形成席夫碱，然后将其减压通过氰基。这种反应dimethylates上的N-末端和赖氨酸侧链和monomethylates N-末端脯氨酸游离氨基。这里所描述的协议甲基化的肽中的样品1使用的试剂与氢原子在其天然同位素分布和样品2使用氘化的甲醛和氰基硼氢化（ 图1）一个"重"标签的"光"的标签。关于在6.0377沓轻质和重质的形式，它是采用利用质谱仪的两种形式之间进行区分之间的质量差异的肽的结果各二甲基化的氨基。具体而言，相对于肽的丰度定量为MS1的提取离子色谱图区域的光的比率（MS1峰面积之比）和重版本的每个肽的离子对。一个蛋白的相对丰度的计算方法中的所有蛋白肽中的中位数MS1峰面积之比。在这份报告中，我们描述了一个强大的协议行为ING雷迪标记实验通过LC-MS/MS，包括反相肽固相萃取，柱上的Redi标签，肽分馏由碱性pH反相（BPRP）层析，并用StageTips肽混合物的纯化（ 图2） 。我们讨论使用REDI标记定量蛋白质组学的优点和局限性。

<table><tbody><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>T9159</td><td>protein precipitation</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>protein precipitation</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>71736</td><td>denature, reduce protein</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td>denature, reduce protein</td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>43816</td><td>denature, reduce, alkylate protein</td></tr><tr><td>Protease Inhibitor Complete Mini Cocktail</td><td>Roche</td><td>4693124001</td><td>denature, reduce protein</td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I6125</td><td>alkylate protein</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H7523</td><td>resuspend, extract, label protein</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C5670</td><td>resuspend protein</td></tr><tr><td>Lysyl Endoprotease</td><td>Wako Chemicals</td><td>129-02541</td><td>protein digestion</td></tr><tr><td>Sequencing grade trypsin</td><td>Promega</td><td>V5111</td><td>protein digestion</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td>protein digestion</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>299537</td><td>Reversed-phase peptide extraction</td></tr><tr><td>tC18 Sep-Pak C18 cartridges</td><td>Waters</td><td>WAT054960</td><td>Reversed-phase peptide extraction</td></tr><tr><td>Extraction manifold</td><td>Waters</td><td>WAT200609</td><td>Reversed-phase peptide extraction</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>14261</td><td>various</td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>252549</td><td>&amp;ldquo;light&amp;rdquo; peptide labeling</td></tr><tr><td>Cyanoborohydride</td><td>Sigma-Aldrich</td><td>71435</td><td>&amp;ldquo;light&amp;rdquo; peptide labeling</td></tr><tr><td>Deuterated formaldehyde</td><td>Sigma-Aldrich</td><td>492620</td><td>&amp;ldquo;heavy&amp;rdquo; peptide labeling</td></tr><tr><td>Sodium cyanoborodeuteride</td><td>CDN isotopes</td><td>D-1797</td><td>&amp;ldquo;heavy&amp;rdquo; peptide labeling</td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td>peptide labeling</td></tr><tr><td>C18-HPLC column (4.6 x 250 mm, 5 &amp;micro;m particle size)</td><td>Agilent</td><td>770450-902</td><td>basic pH reversed-phase chromatography</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>399388</td><td>various</td></tr><tr><td>C18 Empore Disks</td><td>3M</td><td>14-386-3&amp;nbsp;</td><td>STAGE tips</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>494437</td><td>STAGE tips</td></tr></tbody></table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

肽通过还原二甲基化（REDI标签）的稳定同位素标记的方法来准确地量化用质谱样品之间蛋白表达的差异。无论是使用常规（光）或氘（重）形式的甲醛和氰基硼氢化钠加两个甲基对每个游离胺进行REDI标签。在这里，我们展示了一个强大的协议REDI标签和复杂的蛋白质混合物的定量比较。蛋白样品用于比较被消化成肽，标记为运载轻或重的甲基标签，混合，并通过LC-MS/MS共同分析。相对的蛋白丰度，通过比较构成的肽从全MS谱中提取的重链和轻标记版本的离子色谱图的峰面积进行定量。此处描述的方法包括用反相固相萃取的样品制备，肽柱上的Redi贴标，通过碱性pH转肽分馏ersed相（BPRP）层析，并StageTip肽纯化。我们讨论REDI标签的优点和局限性相对于其它方法稳定同位素掺入。我们使用REDI标签作为一种快速，廉价，准确的方法，在几乎任何类型的样品的比较蛋白质丰度突出创新应用。

我们使用酿酒酵母评估的准确度，精密度和雷迪标记的再现性和梭菌C.phytofermentans中全细胞裂解液。我们首先量化C的组合的REDI标记效率C.phytofermentans中的蛋白裂解物纤维素（重标记，H）和葡萄糖（光标签，L）的培养。当过滤，以1％的肽假发现率，该样品含有具有98％的Redi标记效率11194独特的肽序列。未分离S。酵母蛋白溶胞产物同样标记用H或L试剂，混合各种比?...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

肽的还原性二甲基化（REDI标签）稳定同位素标记是一种快速，廉价的战略精确质谱为基础的定量蛋白质组学。这里我们展示了一个可靠的方法用于制备使用可以应用于几乎任何类型的样品中的Redi途径的蛋白质混合物的分析。

定量蛋白质组学使用还原二甲基化的稳定同位素标记

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

quantitative-proteomics-using-reductive-dimethylation-for-stable

Research

JoVE Journal

Chemistry

16.1K 次观看.  Genoscope. 肽的还原性二甲基化（REDI标签）稳定同位素标记是一种快速，廉价的战略精确质谱为基础的定量蛋白质组学。这里我们展示了一个可靠的方法用于制备使用可以应用于几乎任何类型的样品中的Redi途径的蛋白质混合物的分析。 

视频: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

科研

化学

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

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

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

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

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 ...

生物化学

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

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) ...

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

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

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

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

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

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 ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

使用胺还原甲基化进行样品制备和相对定量，用于肽学研究

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

生物学

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

选择反应监测质谱法进行绝对定量蛋白质

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

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