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

11.6K 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 ...

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 ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

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 ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...