We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1.准备裂解液<ol><li>成长P.假单胞菌细胞在LB到所需的外径。 注：对于指导：使用日志阶段文化≈百毫升文化/样品固定相的文化和≈500毫升文化性/样品（OD 600nm的 = 0.5）。 </li><li>粒料通过离心20分钟，在5000×g 下。 </li><li>重悬0.5-1克颗粒在1ml裂解缓冲液（6.7毫MES，6.7毫米的HEPES，200mM的氯化钠，6.7毫醋酸钾，滴滴涕1毫米，pH值7.5）中，并添加蛋白酶抑制剂（完全迷你型，无EDTA），以及作为DNA酶I。 </li><li>裂解的细胞3次传代通过法式压力室，在20,000磅（见材料清单）。 </li><li>超离心细胞裂解物以100,000×g的1小时，在4℃。 </li><li>保存上清液（转到步骤2）。 </li><li>通过上下吹打洗涤用1ml 1X裂解缓冲液沉淀。 </li><li>超速离心机以100,000×g的1小时，在4℃。 </li><li>闪存冻结颗粒在液氮并储存在-20℃直至用于膜蛋白（参见步骤3）的捕获。 </li></ol> 2.去除免费的C-二GMP和其他核苷酸（可溶部分专用） <ol><li>用10ml冷的裂解缓冲液的洗PD10脱盐柱（见材料清单）。 </li><li>倾上清（≈1ml）中到PD10以去除核苷酸。 </li><li>洗脱用4ml冷的裂解缓冲液（500微升步）。 </li><li>选择最集中的分数由Bradford法确定的，集中他们。 </li></ol> 3.颗粒再悬浮和增溶（膜部分只） <ol><li>重悬在500〜1000微升1×捕获缓冲液（无清洁剂）的粒料（ 见表1）。 注：沉淀很难重悬。它应先移液器上下用吸管大致重悬沉淀，然后使用注射器27 G以良好均匀的溶液。 </li><li>添加1％（重量/体积）N-十二烷基β-D-麦芽糖苷（DDM）。 </li><li>孵育在4℃下至少2小时（或O / N）上的旋转轮。 </li><li>超速离心机以100,000×g的1小时，在4℃。 </li><li>收获上清液。 </li></ol> 4.蛋白质浓度测量<ol><li>测量通过Bradford测定法对蛋白浓度（用BCA测定法对膜级分）。 </li><li>设定总蛋白浓度为10毫克/毫升。 </li></ol> 5.捕获<ol><li>混合300微克蛋白质用1mM的GDP，GTP，ATP，CTP和20微升的5×捕获缓冲液（100mM HEPES，250mM的醋酸钾，50mM的MgAc，5％甘油，pH为7.5）（ 表1）。调节反应体积为100微升用H 2 O. 注：总体积包括CDG-CC和c二- GMP的情况下的竞争的控制的体积（见步骤5.3和表2）。 注：所有实验均在200执行＃181; 12升管的PCR条带（Thermo Scientific）进行。 注：对于膜部分，确保始终保持高于临界胶束浓度（0.01％重量/体积），直到在8M尿素的蛋白质溶解步骤（步骤7.1）的DDM浓度。 </li><li>孵育在4℃下进行30分钟，在旋转轮。 </li><li>添加10μM的CDG-CC（最终浓度）。 注：包括但CDG-CC的控制（称为“胎圈”控制），并补充有1mM的C-二叔GMP（“竞赛”控制）的控制（ 见表2）。 注：CDG-CC的浓度可为1至10微米的调整。 </li><li>孵育在4℃下至少2小时（O / N对膜级分）在一个旋转轮，在黑暗中。 </li><li>经过短暂的自旋，交联通过激活CDG-CC的用UV光的反应性部分的4分钟，使用CaproBox（见材料清单，λ= 310纳米，辐照度≥10毫瓦/平方厘米，从源的距离= 2厘米）。注意：删除之前条交联的盖子。 </li><li>加入25微升5倍的洗涤液（5 M氯化钠，250毫米的Tris，pH值7.5）和50微升的再悬浮以及链霉亲和磁珠，轻轻摇匀。 </li><li>孵育在4℃下1小时在旋转轮。 </li></ol> 6.洗涤步骤注：（磁铁：见材料清单）。开始与捕获的在PCR条盖在磁性珠，与磁铁。然后，通过一个新的含有下一洗涤溶液更换的PCR带。取下磁铁和重悬珠，孵育2分钟。降速和新盖更换盖子。 <ol><li>洗涤步骤（可溶部分只） <ol><li>在200μl1×洗涤缓冲液洗涤6次。 </li><li>在200微升HPLC级H 2 O.洗一次</li><li>在200μl80％乙腈洗6次。 </li><li>洗2次在200μlHPLC级H 2 O </li></ol></li><li>洗涤步骤（membraNE分数只） <ol><li>在200μl1×洗涤缓冲液+ 0.1％DDM洗5次。 </li><li>在200μl1×洗涤缓冲液+ 0.05％DDM洗2次。 </li><li>在200微升1×洗涤液+ 0.025％DDM洗一次。 </li><li>在200微升1×洗涤液+ 0.0125％DDM洗一次。 </li><li>在200μl的100mM的ABC洗3次（碳酸氢铵NH 4 CO 3）+ 2M尿素。 </li></ol></li></ol> 7. MS样品制备<ol><li>悬浮珠粒（直接在盖）在20μl100mM的ABC（100毫ABC + 8M尿素的膜级分）并转入1.5毫升管中。 </li><li>膜部分只：孵育在60℃下5分钟，振摇在500rpm。 </li><li>加入0.5微升的200mM TCEP（三（2-羧乙基）膦）和孵育在60℃下1小时，摇动以500rpm。冷却至25℃。 </li><li>加入0.5微升新鲜烹制的400毫米碘乙酰胺和孵化在25℃，30分钟，一个晃动吨500转，在黑暗中。 </li><li>加入0.5微升0.5 M N -乙酰基半胱氨酸和孵育在25℃下进行10分钟，振摇在500rpm。 </li><li>膜组分只有：加1微升赖氨酸-C，37°C，O / N。 </li><li>加入2微克胰蛋白酶和孵化O / N在37°C，震动在500转（包在封口膜，以防止干燥）。 注：样品可以储存在-20℃下，在这个阶段。 注意：膜只能部分：增加100毫米ABC此外胰蛋白酶前调整尿素浓度&lt;2M的。 </li><li>简要降速管和收集珠磁铁。 </li><li>转移上清液到新的1.5毫升管（重复此步骤，如果小珠留）。 </li><li>添加5微升5％的TFA（三氟乙酸）+1微升2 M盐酸（15微升5％的TFA + 5微升的2M HCl中的膜部分）。 </li><li>条件的C18 MicroSpin列（巢组，MA，USA）用150微升的乙腈（旋涂20秒，在2400转）。 </li><li>球菌保持平衡的C18柱2次用150微升0.1％TFA（自旋20秒，2400转）。 </li><li>加载样品和自旋2分钟，2000转。 </li><li>重载流通到柱并重复纺丝步骤（2分钟，2000转）。 </li><li>洗3次，用150微升0.1％的TFA，5％乙腈（20秒，2400转）。 </li><li>取一个新的试管中，并用150微升0.1％TFA洗脱两次，50％乙腈（2分钟，2000转）。 </li><li>干燥的肽在Speed​​-Vac中。 </li><li>重悬在40微升98％的H 2 O，2％乙腈，0.15％甲酸。 </li><li>超声处理20秒（脉冲周期0.5，幅度100％;见材料清单）和自旋向下5秒，12000转（台式离心机）。旋涡10秒，降速5秒，12000转。转移在HPLC小瓶用于LC-MS / MS分析。 </li><li>冷冻在-20℃。 注：样品可以储存在-20℃下，在这个阶段。 </li></ol> 8. LC-MS / MS分析<ol><li>运行纳米LC（纳米LC系统）equipp编带型RP-HPLC柱（75微米×37厘米）填充有C18的树脂（魔术C18 AQ 3微米），使用线性梯度从95％溶剂A（0.15％甲酸，2％乙腈）和5％溶剂B（ 98％乙腈，0.15％甲酸）至35％溶剂B在60分钟内以0.2微升/分钟的流速。 </li><li>分析用LC-MS / MS（双重压力LTQ-的Orbitrap Velos的质谱仪，连接到电喷雾离子源）的肽。 注意：数据采集模式被设置以获得1个高分辨率的MS质谱仪中的FT部在60,000 FWHM的分辨率随后的MS / MS扫描中的20个最激烈的离子的线性离子阱扫描。以增加的MS / MS尝试的效率，充电状态的筛选工作方式被启用以排除未分配和单独充电的离子。勾结诱导解离被触发时，前体超过100离子计数。动态排除持续时间为30秒。离子累积时间设定为300毫秒（MS）和50毫秒（MS / MS）。 </li></ol> 9.数据库搜索<ol><li>下载P.铜绿假单胞菌 NCBI数据库通过NCBI主页（ <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ）。 </li><li>转换使用MassMatrix转换工具（MS原谱成吉祥物通用文件（MGF） <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ）。 </li><li>使用MASCOT 2.3版本对P.搜索此MGF文件假单胞菌 NCBI数据库含有正向和反向诱饵蛋白的条目。 </li><li>赖氨酸和精氨酸（除非其次是脯氨酸）容忍2错过了完全胰蛋白酶肽分裂后执行在硅片胰蛋白酶消化。 </li><li>设置数据库的搜索参数，使氧化蛋氨酸（15.99491大）为变量修改和半胱氨酸残基固定修改carboxyamidomethylation（57.021464大）。对于MASCOT我们搜索荷兰国际集团的高分辨率扫描，设置的前体质量耐受至15ppm，并设置碎片质量公差到0.6沓。最后，设置蛋白FDR至1％。 </li><li>进口P.的吉祥物搜索绿脓杆菌 CCMS实验到脚手架（Proteomesoftware，第3版），设置参数，以获得蛋白质FDR接近1％，并提取总光谱计数。 </li><li>为代表性结果呈现在本文中，我们使用了配对T检验的实验与竞争控制比较，并只考虑命中低于0.1 的 p值，和上述2（实验光谱计数/竞争控制光谱的光谱计数比计数）。 </li><li>数据可在任何电子表格软件进行进一步的分析来导出。 </li></ol> 10.无标签定量<ol><li>导入原始文件到Progenesis LC-MS软件（非线性动力学，4.0版）。 </li><li>执行LC-MS校准和特征检测的默认settiNGS。 </li><li>从Progenesis LC-MS导出MGF格式的数据。 </li><li>使用搜索对NCBI P.吉祥物的MS / MS谱含有正向和反向诱饵蛋白条目假单胞菌数据库。 </li><li>导入数据库搜索结果转换成Progenesis LC-MS和映射的肽鉴定到MS1特征。 </li><li>对于数据评估（显着性水平计算，折换率）SafeQuant的ProteinSQAnalysis脚本使用（阈值：低于0.1的Q值，高于2谱计数率）。 </li></ol>

<ol>
	<li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> J Proteomics. 75, 4874-4878 (2012).</li><li>Hengge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+c-di-GMP+signalling+in+bacteria.">Principles of c-di-GMP signalling in bacteria.</a> Nature reviews. Microbiology. 7, 263-273 (2009).</li><li>Sondermann, H., Shikuma, N. J., Yildiz, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=You've+come+a+long+way:+c-di-GMP+signaling.">You've come a long way: c-di-GMP signaling.</a> Curr. Opin. Microbiol. 15, 140-146 (2012).</li><li>Schirmer, T., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+mechanistic+determinants+of+c-di-GMP+signalling.">Structural and mechanistic determinants of c-di-GMP signalling.</a> Nature reviews. Microbiology. 7, 724-735 (2009).</li><li>Furukawa, S., Kuchma, S., O'Toole, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+their+options+open:+acute+versus+persistent+infections.">Keeping their options open: acute versus persistent infections.</a> J. Bacteriol. 188, 1211-1217 (2006).</li><li>Christen, M., Christen, B., Folcher, M., Schauerte, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+cyclic+di-GMP-specific+phosphodiesterase+and+its+allosteric+control+by+GTP.">Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP.</a> J. Biol. Chem. 280, 30829-30837 (2005).</li><li>Ryan, R. P., Fouhy, Y., Lucey, J. F., Dow, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-GMP+signaling+in+bacteria:+recent+advances+and+new+puzzles.">Cyclic di-GMP signaling in bacteria: recent advances and new puzzles.</a> J. Bacteriol. 188, 8327-8334 (2006).</li><li>Habazettl, J., Allan, M. G., Jenal, U., Grzesiek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+structure+of+the+PilZ+domain+protein+PA4608+complex+with+cyclic+di-GMP+identifies+charge+clustering+as+molecular+readout.">Solution structure of the PilZ domain protein PA4608 complex with cyclic di-GMP identifies charge clustering as molecular readout.</a> J. Biol. Chem. 286, 14304-14314 (2011).</li><li>Fazli, M., 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+CRP/FNR+family+protein+Bcam1349+is+a+c-di-GMP+effector+that+regulates+biofilm+formation+in+the+respiratory+pathogen+Burkholderia+cenocepacia.">The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia.</a> Molecular Microbiology. 82, 327-341 (2011).</li><li>Hickman, J. W., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FleQ+from+Pseudomonas+aeruginosa+as+a+c-di-GMP-responsive+transcription+factor.">Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor.</a> Molecular Microbiology. 69, 376-389 (2008).</li><li>Sudarsan, N., 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=Riboswitches+in+eubacteria+sense+the+second+messenger+cyclic+di-GMP.">Riboswitches in eubacteria sense the second messenger cyclic di-GMP.</a> Science. 321, 411-413 (2008).</li><li>Lenz, T., 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=Profiling+of+methyltransferases+and+other+S-adenosyl-L-homocysteine-binding+Proteins+by+Capture+Compound+Mass+Spectrometry+(CCMS).">Profiling of methyltransferases and other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS).</a> J Vis Exp. , (2010).</li><li>K&#246;ster, H., 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+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev Technol. 5, 381-390 (2007).</li><li>D&#252;vel, 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=A+chemical+proteomics+approach+to+identify+c-di-GMP+binding+proteins+in+Pseudomonas+aeruginosa.">A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.</a> Journal of Microbiological Methods. 88, 229-236 (2012).</li><li>Christen, 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=Allosteric+control+of+cyclic+di-GMP+signaling.">Allosteric control of cyclic di-GMP signaling.</a> J. Biol. Chem. 281, 32015-32024 (2006).</li><li>Balasubramanian, D., Mathee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptome+analyses+of+Pseudomonas+aeruginosa.">Comparative transcriptome analyses of Pseudomonas aeruginosa.</a> Human genomics. 3, 349-361 (2009).</li><li>Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+models+and+empirical+bayes+methods+for+assessing+differential+expression+in+microarray+experiments.">Linear models and empirical bayes methods for assessing differential expression in microarray experiments.</a> Stat Appl Genet Mol Biol. 3 (3), (2004).</li><li>Baraquet, C., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+diguanosine+monophosphate+represses+bacterial+flagella+synthesis+by+interacting+with+the+Walker+A+motif+of+the+enhancer-binding+protein+FleQ.">Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 18478-18483 (2013).</li><li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> J Proteomics. 75, 4874-4878 (2012).</li><li>Roelofs, K. G., Wang, J., Sintim, H. O., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+for+high-throughput+detection+of+protein-metabolite+interactions.">Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 15528-15533 (2011).</li><li>DeSantis, K., Reed, A., Rahhal, R., Reinking, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+differential+scanning+fluorimetry+as+a+high-throughput+assay+to+identify+nuclear+receptor+ligands.">Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.</a> Nuclear receptor signaling. 10, e002 (2012).</li><li>Seidel, S. A., 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=Microscale+thermophoresis+quantifies+biomolecular+interactions+under+previously+challenging+conditions.">Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.</a> Methods. 59, 301-315 (2013).</li><li>Merighi, M., Lee, V. T., Hyodo, M., Hayakawa, Y., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+messenger+bis-(3&#8216;-5&#8217;)-cyclic-GMP+and+its+PilZ+domain-containing+receptor+Alg44+are+required+for+alginate+biosynthesis+in+Pseudomonas+aeruginosa.">The second messenger bis-(3&#8216;-5&#8217;)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa.</a> Molecular Microbiology. 65, 876-895 (2007).</li><li>Qi, 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=Binding+of+cyclic+diguanylate+in+the+non-catalytic+EAL+domain+of+FimX+induces+a+long-range+conformational+change.">Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change.</a> J. Biol. Chem. 286, 2910-2917 (2011).</li></ol>

The authors have nothing to disclose.

特别应注意，在几个步骤的协议。蛋白质浓度是用10毫克的浓度的关键参数/ ml的暂时难以达到当细胞在特定的生长条件（ 例如生物膜或小菌落变体）生长。因此，将沉淀再悬浮应在裂解缓冲液的低体积来进行。蛋白质浓度可以降低到8毫克/毫升。相比由Nesper 等人的 1公开的方法中，我们加入不同的核苷酸的捕获反应，以尽量减少核苷酸结合蛋白的非特异性捕获。虽然在加入?...

C-二叔GMP的是所使用的大多数细菌，以控制它们的生长和行为的各种方面的关键的第二信使。例如，C-二叔GMP的调节细胞周期进程，迁移和胞外多糖和表面粘附2-4的表达。通过这样的过程的协调的c-二叔GMP的促进生物膜的形成，这是与一个范围病原菌5的慢性感染相关的处理。 C-二GMP是由酶称为diguanylate环化酶（DGCS）窝藏催化GGDEF域4合成出。一些DGCS具有抑制网站在C-二GMP绑定下调的环化酶活性。 C-二叔GMP的降解是由两个不同的类磷酸二酯酶（PDE）窝藏或者催化EAL或HD-GYP域6,7催化。 大部分已知的效应蛋白的直接结合的c-二叔GMP仅属于三类蛋白之一：催化盟友不活跃GGDEF或EAL域和域皮尔磁，即发生在C-二GMP结合8的构象变化小的分子开关。 DGCS，偏微分方程和皮尔磁蛋白很好的特点和他们的域可以相对安全地预测，在硅片 。特别感兴趣，现在专注于新型的C-二GMP效应的鉴定。一些C二叔GMP的效应具有不同结合基序被描述最近诸如CRP / FNR蛋白家族Bcam1349在伯克霍尔德新洋葱伯克霍尔德杆菌或P的转录调节FLEQ 铜绿假单胞菌 9,10。此外，C-二叔GMP特异性核糖开关最近确定和示出，以控制基因表达的c二叔GMP依赖性11。不同效应的c二叔GMP的结合基序仅很差保守制造这种蛋白质的生物信息学预测困难。为了解决这个问题，我们开发了一种生物化学方法，它是基于使用一个C二叔GMP的SPE的cific捕捉化合物结合质谱1,12,13。 我们最近设计了一种新的三价的c-二叔GMP的捕捉化合物（CDG-CC中， 图1）1。这种化学物质的支架的组成如下：1）一个用作诱饵捕获的c-二叔GMP的结合蛋白的c-二叔GMP的部分，2）紫外线光活化反应性基团用于交联所述CDG-CC的结合的蛋白质和3）生物素分离使用链霉亲和包被的磁珠捕获的蛋白质。在CDG-CC可用于直接和特异性从大分子作为细胞裂解物的复杂混合物捕获的c-二叔GMP的效应。捕获复合基和化学基蛋白质组学方法先前已报道为适用于广泛范围的生物， 例如柄杆菌新月 ， 沙门鼠伤寒和P.铜绿假单胞菌 1,14。 在这种方法的论文，我们提供了一个使用P的提取CCMS程序的深入介绍假单胞菌作为一个例子。本研究建立CCMS作为一个强大和灵活的工具，生化鉴定涉及小分子信号的新组件。

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

在对第二信使C-二GMP的参与合成（diguanylate环化酶）和降解（磷酸二酯酶）酶的鉴定和表征过去的十年里已经取得了长足的进步。与此相反，小的信息是可用的关于分子机制和细胞成分通过此信号传导分子调节的细胞过程的一个不同的范围。大多数已知的效应蛋白属于Pilz家族或正在退化diguanylate环化酶和磷酸二酯酶已经放弃了催化作用，并已采取效应功能。因此，为了在大范围细菌的实验方法是必需的，以确定和验证的量可靠，在硅片预测失败新颖效应的更好地确定蜂窝的c-二叔GMP的网络。 我们最近开发出一种新型的复合捕获质谱（CCMS）基于技术作为一个强大的工具，生化识别和描述的c-二GMP结合蛋白。这种技术先前已经报道为适用于广泛范围的生物体1。在这里，我们给大家利用来探测这样的信号成分的协议的详细说明。作为一个例子，我们使用绿脓杆菌 ，一种条件致病菌其中c二叔GMP起着毒力和生物膜控制的关键作用。 CCMS确定的c二叔GMP的网络的已知或预测的部件的74％（38/51）。这项研究详细解释了CCMS程序，并建立它作为一个强大和灵活的工具，以查明参与小分子信号的新组件。

为了找出P.新颖C-二GMP效应铜绿假单胞菌 ，我们系统地使用CCMS分析P.的可溶性和膜组分从对数期培养假单胞菌菌株PAO1（OD 600 = 0.5）。在这里，我们总结和讨论这个摸底的代表性结果。四个独立的生物复制品被使用。对于每个实验两个不同CDG-CC的浓度使用（5微米和10微米）。探测特异性，进行了实验，在1mM的C-二叔GMP作为竞争，最后在存在或不存在，用珠对照（ <e...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K 次观看.  Biozentrum of the University of Basel. The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

视频: Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

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 ...