作者感谢肯特西利，该中心药物发现与创新（CDDI）蛋白质组学核心设施在南佛罗里达大学使用该设施的主任医师。这项工作是由美国国立卫生研究院/ NIDCD授予R01 DC004295到BHAS支持

操守准则利用小鼠体内实验已获南佛罗里达大学实验动物管理和使用委员会（协议3931R，3482R）作为载下美国国立卫生研究院的指导方针。 1。蛋白抽提<ol><li>在-80°C隔离耳蜗感觉上皮从16 30日龄（P30）CBA / J小鼠和存储</li><li>在实验当天，用500μl1X磷酸盐缓冲盐水（PBS）中洗涤组织。离心3分钟，在1000×g离心，去除上清。重复总共3次洗涤。 </li><li> 30秒在冰上于含有50mM的Tris-HCl，pH值8.0，120 mM氯化钠，50mM的氟化钠，5mM的EDTA，500微克/毫升AEBSF，10微克/毫升亮肽素，100微克/ 100微升裂解缓冲液中超声处理组织毫升胃蛋白酶，2微克/毫升抑肽酶和0.5微克/毫升的微囊藻毒素使用声波dismembrator（型号100，赛默飞世尔）。冰酷裂解液每个超声处理之间1分钟。超声处理共3倍的。 </li><li>离心提取物，在750×g离心在4℃下持续2分钟，去除上清至新的微管中。通过超声处理30秒，在冰上提取沉淀中加入50μl裂解缓冲液。离心提取物，在750×g离心在4℃下持续2分钟。在28,600 XG结合这两种裂解液和离心机在4℃下60分钟。除去上清液至新的微管中，并加入含0.1％ASB-14至沉淀20微升裂解缓冲液。涡旋混合1分钟，孵育60分钟，在4℃下</li><li>加热该样品在95℃下5分钟，然后冷却，在4℃下进行60分钟。用离心跟随在16000 XG在25°C下10分钟。收集上清液并转移到新管中。 </li><li>离心悬浮液以16,000×g离心在4℃下5分钟，并保留上清液进行消化。 </li></ol> 2。全裂解液的使用FASP双胰蛋白酶消化蛋白质<ol><li>添加30μ耳蜗蛋白提取物，含有4％十二烷基硫酸钠（SDS）的升等分试样（≤400微克），100mM的Tris-盐酸，pH值7.6和0.1M二硫苏糖醇（DTT）直接到30K自旋过滤器和混合用200μl的8尿素的Tris-HCl中。离心机以14,000×g离心15分钟。 </li><li>稀释该浓缩物用200μl的在14,000 xg离心15分钟，8M尿素溶液和离心机。 </li><li>在8M尿素溶液中，以该浓缩物在过滤器中并涡旋混合1分钟加入10μl的10倍碘乙酰胺（IAA）。孵育自旋过滤器在室温（RT）下在黑暗中20分钟，然后离心，14,000 xg离心10分钟。 </li><li>加入100微升的8M尿素溶液的浓缩物以14,000×g离心15分钟，过滤器单元和离心分离机上。重复此步骤2倍。加入100微升的50mM的碳酸氢铵（ABC）的溶液于14,000 xg离心10分钟，过滤器单元和离心分离机。重复此步骤2倍。 </li><li>添加0.4微克/微升的胰蛋白酶在1:100（W​​ / W）的酶，以蛋白质比例孵育过夜（O / N）在37°C </li><li>温育后，加入40微升50mM的ABC溶液过滤单元和离心机以14,000×g离心10分钟。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液以14,000×g离心10分钟自旋过滤器和离心机。含有胰蛋白酶肽的滤液转移到一个新的微管中，用甲酸（FA）酸化至1.0％，以终止消化。 </li><li>用40微升8M尿素冲洗过滤器单元，然后用40微升18兆欧水清洗2倍。 </li><li>用100μl50mM的ABC溶液冲洗过滤器单元3倍。经过最后一次洗涤加0.4微克/微升胰蛋白酶1:100（W​​ / W）的酶与蛋白质的比例和孵化O / N为37°C。 </li><li>通过加入40微升50mM的ABC溶液以14,000×g离心，过滤器单元和离心分离10分钟，洗脱胰蛋白酶肽从第二消化。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液对以14,000×g离心10分钟过滤器单元和离心分离机。含有胰蛋白酶肽的滤液转移到一个新的微管，并与FA酸化到1.0％。 </li><li>样品可以使用牛血清白蛋白作为标准，使用微孔板比色测定法进行定量。 </li></ol> 3。全裂解液的使用FASP内蛋白酶LYSC和胰蛋白酶消化蛋白质<ol><li>添加的含有4％SDS，100mM的Tris-盐酸，pH值7.6和0.1 M DTT直接到30K自旋过滤器耳蜗蛋白提取物的30微升等分试样（≤400微克），并用200μl的8M尿素中的Tris-HCl混合。离心机以14,000×g离心15分钟。 </li><li>稀释该浓缩物用200μl的在14,000 xg离心15分钟，8M尿素溶液和离心机。 </li><li>加入10μl的10倍IAA在8M尿素溶液中，以该浓缩物在过滤器中并涡旋混合1分钟。孵育自旋过滤器在室温20分钟，在黑暗中，然后离心，14,000 xg离心10分钟。 </li><li>加入100μl的8M尿素溶胶ution到该浓缩物以14,000×g离心15分钟，过滤器单元和离心分离机上。重复此步骤2倍。加入100微升100mM的ABC溶液以14,000×g离心10分钟，过滤器单元和离心分离机。重复此步骤2倍。 </li><li>添加0.1微克/微升内蛋白酶LYSC在1:50（W / W）的酶与蛋白质的比例和孵化O / N为30°C。 </li><li>温育后，加入40微升100mM ABC溶液以14,000×g离心10分钟，过滤器单元和离心分离机。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液以14,000×g离心10分钟自旋过滤器和离心机。含LYSC肽滤液转移到一个新的微管，并与FA酸化到1.0％，停止消化。 </li><li>用40微升8M尿素冲洗过滤器单元，然后用40微升18兆欧水清洗2倍。 </li><li>用100μl50mM的ABC溶液冲洗过滤器单元3倍。后在最后一次洗涤中添加0.4微克/微升的胰蛋白酶在1:100的酶对亲蛋白质比例和孵化O / N为37°C。 </li><li>通过加入40微升50mM的ABC溶液以14,000×g离心10分钟，过滤器单元和离心分离机中洗脱胰蛋白酶肽。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液，以在14,000×g离心10分钟，过滤器单元和离心分离机。含有胰蛋白酶肽的滤液转移到一个新的微管和酸化用1.0％FA。 </li><li>样品可以用微孔板比色法用BSA为标准进行定量。 </li></ol> 4。脱盐肽使用离心柱<ol><li>通过加入500μl的乙腈（ACN）和离心机在1100×g离心1分钟，激活一个C 18 MacroSpin列。离心后丢弃的流量通过。 </li><li>用500μl0.1％甲酸和离心机的平衡色谱柱在1100×g离心1分钟。丢弃的流量通过并重复此步骤1X。 </li><li>最多可添加500微升肽的消化到列的ð离心机在1100×g离心1分钟。如果样品体积大于500微升然后重复此步骤。 </li><li>用500微升0.1％FA和离心机在1100×g离心1分钟洗柱。弃去流过。重复此步骤1X。 </li><li>加入250μl的90:10的乙腈 - 水比例在1100×g离心1分钟，柱和离心机。收集含有脱盐肽洗脱液，转移到一个新的微管。重复此步骤1X。 </li><li>干燥脱盐肽样品在真空离心机和完全避免让样品干燥。 </li></ol> 5。离子交换色谱<ol><li>注入50-100消化蛋白质样品微克到200×2.1毫米，5微米SCX柱（聚磺A）分离肽。 </li><li>使用2-40％B，用50分钟的梯度为250微升/分钟的流速。溶剂A为5 mM甲酸铵，pH值3.0在25％乙腈和75％的双蒸2 O。溶剂B为500 mM甲酸铵，pH值6.0在25％乙腈和75％的双蒸2 O。 </li><li>监视肽组分在280nm处，并用流分收集器收集在2分钟间隔的级分。 </li><li>干燥的部分在真空浓缩，重悬在500微升50％乙腈在DDH 2 O含5％FA协助盐去除。 </li><li> Redry分数和储存在-80°C，直到准备使用纳米LC-MS/MS分析。 </li></ol> 6。丙酮沉淀前GELFrEE分离耳蜗蛋白的上清液，必须脱盐。丙酮沉淀法可用于脱盐和浓缩的蛋白质。 <ol><li>添加三卷冰冷的丙酮耳蜗上清液。轻轻振荡后，在-20°CO / N。 </li><li>以15,000×g离心离心样品混合物15分钟，在4℃下，去除上清。 </li><li>用冷丙酮和离心机在14,000 XG在4清洗蛋白沉淀3次5分钟°C。 </li><li>空气干燥沉淀，溶解于112微升的18MΩ水。 </li><li>使用酶标仪比色法测定蛋白质浓度。 </li></ol> 7。人工耳蜗感觉上皮的GELFrEE分馏<ol><li>加入30微升的5×样品缓冲液（0.25M的Tris-HCl pH 6.8的10％（重量/体积）SDS，50％甘油，0.5％（重量/体积）溴酚蓝）的脱盐的蛋白质悬浮在112微升18MΩ水。 </li><li>添加8微升1M的DTT和热量在95℃下5分钟，以减少蛋白质二硫键。加热后允许冷却到室温。 </li><li>加8毫升运行缓冲液，以阳极槽，150微升缓冲液样品收集室的，和6ml缓冲液至阴极贮存。 </li><li>除去可能已使用移液管流入试样装填室的任何缓冲液。 </li><li>负载耳蜗蛋白质混合物的150微升（≤1毫克），进入试样装填室使用8％蒸腾是醋酸盒和3.5-150 kDa的质量范围。 </li><li>下电极和关闭仪器盖子开始运行。 </li><li>在第一次暂停的时间在仪器上加入2ml缓冲液的阴极槽和恢复运行。 </li><li>在仪器上的每一暂停的时间间隔，用移液管收集来自样本的收集腔室中的样品，并加入到一个新鲜标记的微管中。用150微升运行缓冲液冲洗收集室2倍，并丢弃。 </li><li>加入150μl新鲜运行缓冲液的样品收集腔，恢复运行。收集的蛋白组分过2.6小时，在150微升/分的总体积的时间段。 </li></ol> 8。 GELFrEE分数的1D凝胶电泳 1D凝胶电泳可用于可视化之前的酶消化和质谱分析结果从GELFrEE分馏。 GELFrEE蛋白组分可以分离在4-15％的Tris-HCl的凝胶。 </p&gt; <ol><li>混合每GELFrEE部分的5微升等份与5微升的样品稀释缓冲液（350 mM的DTT在样品缓冲液）。 </li><li>加热该样品在95℃下3分钟。 </li><li>凉爽的样品至室温。 </li><li>负载10微升每个GELFrEE分数在个别胶道和负载5微升的最后一个未使用的车道分子量标准。 </li><li>运行该凝胶在125 V 1.5小时或直至染料前沿到达凝胶的底部。 </li><li>小心取出凝胶，并用银染染色加上可视化的蛋白质分离。 </li></ol> 9。使用专上学生资助GELFrEE部分的蛋白质消化一种改性FASP程序是用于洗涤剂除去GELFrEE馏分和消化。 <ol><li>每个单独的馏分直接添加到30K滤波器单元;用200μl8M尿素的Tris-HCl和离心机以14,000×g离心25分钟混合。 </li><li>稀释该浓缩物用200μl的尿素溶液和离心机以14,000×g离心12分钟。重复此步骤1X。 </li><li>加入10μl的10倍IAA在每个滤波器单元中，涡旋混合1分钟尿素溶液浓缩物中，并孵育在室温下30分钟，在黑暗中。 </li><li>加入100微升的尿素溶液以14,000×g离心15分钟，将浓缩物在过滤器单元和离心分离机。重复此步骤2倍。加入100微升100mM的ABC溶液至过滤器单元和离心机以14,000×g离心10分钟。重复此步骤2倍。 </li><li>为1:50（W / W）的酶与蛋白质之比加入0.1微克/微升的lysC的向过滤器单元和孵育O / N在30℃下</li><li>温育后，加入40微升100mM ABC溶液的自旋过滤器和离心机以14,000×g离心10分钟。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液，以自旋过滤器和离心机以14,000×g离心10分钟。包含从每个自旋过滤器的LYSC肽滤液转移到一个新的微管和酸化与trifluoroac客位乙酸（TFA）。 </li><li>用40微升8M尿素洗涤离心过滤，然后用40微升18兆欧水清洗2倍。 </li><li>洗自旋过滤器3倍用100μl50mM的ABC溶液。经过最后一次洗涤加0.4微克/微升胰蛋白酶在1:100（W​​ / W）的酶与蛋白质的比例和孵化O / N为37°C。 </li><li>加入40微升的50毫米农行的解决方案和离心14,000×g离心10分钟洗脱肽段从每个过滤单元。重复此步骤1X。 </li><li>加入50微升0.5M的NaCl溶液的过滤器单元和离心机以14,000×g离心10分钟。包含从每个过滤单元胰蛋白酶肽的滤液转移到一个新的微管和酸化与TFA。 </li><li>干全部精华在真空浓缩。 </li></ol> 10。样品制备LC-MS/MS <ol><li>重组干LYSC和胰蛋白酶消化肽组分在20微升0.1％FA和旋涡。 </li><li>离心样品以20,000×g离心10分钟，除去顶部95％的样本的一个新的样品瓶。 </li><li>注入5μl的每种肽馏分到100微米×25毫米的样品阱，以除去盐和污染物，并以75微米×10厘米-C 18柱进行色谱分离。 </li><li>使用2-40％B，历经100分钟的梯度为200升/分钟的流速。溶剂A为95％，双蒸水和5％乙​​腈含0.1％FA。溶剂B为80％乙腈和20％的双蒸水含0.1％FA。 </li><li>收集10串联质谱对每个MS扫描的高分辨率质谱仪。一个LTQ的Orbitrap质谱仪被用于本实验。 </li></ol> 11。蛋白质鉴定<ol><li>使用包含的UniProt数据库鼠标向前识别蛋白质和反向蛋白序列和常见污染物。与福星搜索引擎MaxQuant软件是用来搜索蛋白质数据库。 </li><li>搜索参数，可用于包括;固定modific半胱氨酸的脲基甲基的振动性，蛋氨酸的氧化变量的修改，蛋白质N-末端乙酰化，最大为2遗漏乳沟。最小肽的长度如何考虑的识别是6个氨基酸。 输入±8 ppm或1.2大（质量误差是依赖于所使用的质谱仪）的片段质量误差肽质量浓度的错误。 </li><li>在MaxQuant软件，用1％的假发现率（FDR）的肽和蛋白质鉴定。蛋白质鉴定中列出具有匹配的肽，该蛋白的百分比序列覆盖率，并且所述肽序列的数量。 </li></ol>

<ol>
	<li>Domon, B., 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=Review+-+Mass+spectrometry+and+protein+analysis.">Review - Mass spectrometry and protein analysis.</a> Science. 312, 212-217 (2006).</li><li>Jamesdaniel, S., Hu, B., Kermany, M. H., Jiang, H., Ding, D., Coling, D., Salvi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+induced+changes+in+the+expression+of+p38/MAPK+signaling+proteins+in+the+sensory+epithelium+of+the+inner+ear.">Noise induced changes in the expression of p38/MAPK signaling proteins in the sensory epithelium of the inner ear.</a> J. Proteomics. 75, 410-424 (2011).</li><li>Thalmann, I., Hughes, I., Tong, B. D., Ornitz, D. M., Thalmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+analysis+of+proteins+in+inner+ear+tissues+and+fluids+with+emphasis+on+endolymphatic+sac,+otoconia,+and+organ+of+Corti.">Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti.</a> Electrophoresis. 27, 1598-1608 (2006).</li><li>Thalmann, I., Rosenthal, H. L., Moore, B. W., Thalmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+of+Corti-specific+polypeptides:+OCP-I+and+OCP-II.">Organ of Corti-specific polypeptides: OCP-I and OCP-II.</a> J. Acoust. Soc. Am. 67, (1980).</li><li>Kathiresan, T., Harvey, M., Sokolowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The use of two-dimensional gels to identify novel protein-protein interactions in the cochlea. In Auditory and vestibular research : methods and protocols. , (2009).</li><li>Zheng, Q. Y., Rozanas, C. R., Thalmann, I., Chance, M. R., Alagramam, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+proteomics+of+mouse+models+for+deafness,+a+discovery+strategy.">Inner ear proteomics of mouse models for deafness, a discovery strategy.</a> Brain Res. 1091, 113-121 (2006).</li><li>Peng, H., Liu, M., Pecka, J., Beisel, K. W., Ding, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+the+Organ+of+Corti+Using+Nanoscale+Liquid+Chromatography+Coupled+with+Tandem+Mass+Spectrometry.">Proteomic Analysis of the Organ of Corti Using Nanoscale Liquid Chromatography Coupled with Tandem Mass Spectrometry.</a> Int. J. Mol. Sci. 13, 8171-8188 (2012).</li><li>Elkan-Miller, T., Ulitsky, I., Hertzano, R., Rudnicki, A., Dror, A. A., Lenz, D. R., Elkon, R., Irmler, M., Beckers, J., Shamir, R., Avraham, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+Transcriptomics,+Proteomics,+and+MicroRNA+Analyses+Reveals+Novel+MicroRNA+Regulation+of+Targets+in+the+Mammalian+Inner+Ear.">Integration of Transcriptomics, Proteomics, and MicroRNA Analyses Reveals Novel MicroRNA Regulation of Targets in the Mammalian Inner Ear.</a> Plos One. 6, (2011).</li><li>Yang, Y., Dai, M., Wilson, T. M., Omelchenko, I., Klimek, J. E., Wilmarth, P. A., David, L. L., Nuttall, A. L., Gillespie, P. G., Shi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+/K+-ATPase+&#945;1Identified+as+an+Abundant+Protein+in+the+Blood-Labyrinth+Barrier+That+Plays+an+Essential+Role+in+the+Barrier+Integrity.+.">Na+/K+-ATPase &#945;1Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity. .</a> Plos One. 6, (2011).</li><li>Shin, J. B., Streijger, F., Beynon, A., Peters, T., Gadzala, L., McMillen, D., Bystrom, C., Vander Zee, C. E., Wallimann, T., Gillespie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+Bundles+Are+Specialized+for+ATP+Delivery+via+Creatine+Kinase.">Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.</a> Neuron. 53, 371-386 (2007).</li><li>Chen, Z. Y., Corey, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inner+ear+gene+expression+database.">An inner ear gene expression database.</a> J. Assoc. Res. Otolaryngol. 3, 140-148 (2002).</li><li>Gabashvili, I. S., Sokolowski, B. H., Morton, C. C., Giersch, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+gene+expression+in+the+inner+ear.">Ion channel gene expression in the inner ear.</a> J. Assoc. Res. Otolaryngol. 8, 305-328 (2007).</li><li>Horvatovich, P., Hoekman, B., Govorukhina, N., Bischoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+chromatography+coupled+to+mass+spectrometry+in+analysing+complex+proteomics+samples.">Multidimensional chromatography coupled to mass spectrometry in analysing complex proteomics samples.</a> J. Sep. Sci. 33, 1421-1437 (2010).</li><li>Ye, M. L., Jiang, X. G., Feng, S., Tian, R. J., Zou, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+chromatographic+techniques+and+methods+in+shotgun+proteome+analysis.">Advances in chromatographic techniques and methods in shotgun proteome analysis.</a> Trends Anal. Chem. 26, 80-84 (2007).</li><li>Wu, C. C., MacCoss, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+proteomics:+Tools+for+the+analysis+of+complex+biological+systems.">Shotgun proteomics: Tools for the analysis of complex biological systems.</a> Curr. Opin. Mol. Ther. 4, 242-250 (2002).</li><li>Bora, A., Anderson, C., Bachani, M., Nath, A., Cotter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Two-Dimensional+Separation+of+Intact+Proteins+for+Bottom-Up+Tandem+Mass+Spectrometry+of+the+Human+CSF+Proteome.">Robust Two-Dimensional Separation of Intact Proteins for Bottom-Up Tandem Mass Spectrometry of the Human CSF Proteome.</a> J. Proteome Res. 11, 3143-3149 (2012).</li><li>Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry:+Bottom-up+or+top-down.">Mass spectrometry: Bottom-up or top-down.</a> Science. 314, 65-66 (2006).</li><li>Aebersold, R., 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=Mass+spectrometry-based+proteomics.">Mass spectrometry-based proteomics.</a> Nature. 422, 198-207 (2003).</li><li>Reid, G. E., McLuckey, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+down'+protein+characterization+via+tandem+mass+spectrometry.">Top down' protein characterization via tandem mass spectrometry.</a> J. Mass. Spectrom. 37, 663-675 (2002).</li><li>Han, X., Aslanian, A., Yates, 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=Mass+spectrometry+for+proteomics.">Mass spectrometry for proteomics.</a> Curr. Opin. Chem. Biol. 12, 483-490 (2008).</li><li>Thalmann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+ear+proteomics:+A+fad+or+hear+to+stay.">Inner ear proteomics: A fad or hear to stay.</a> Brain Res. 1091, 103-112 (2006).</li><li>Lang, F., Vallon, V., Knipper, M., Wangemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+channels+and+transporters+expressed+in+the+inner+ear+and+kidney.">Functional significance of channels and transporters expressed in the inner ear and kidney.</a> Am. J. Physiol. Cell Physiol. 293, (2007).</li><li>Wisniewski, J. R., Zougman, A., Nagaraj, N., 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=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nat. Meth. 6, 359-362 (2009).</li><li>Wisniewski, J. R., Zielinska, D. F., 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=Comparison+of+ultrafiltration+units+for+proteomic+and+N-glycoproteomic+analysis+by+the+filter-aided+sample+preparation+method.">Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method.</a> Anal. Biochem. 410, 307-309 (2011).</li><li>Darville, L. N., Sokolowski, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+Proteomic+Analysis+of+Mouse+Cochlear+Sensory+Epithelium+by+Mass+Spectrometry.">In-depth Proteomic Analysis of Mouse Cochlear Sensory Epithelium by Mass Spectrometry.</a> J Proteome Res. 12 (8), 3620-3630 (2013).</li><li>Vizcaino, J. A., Cote, R. G., Csordas, A., Dianes, J. A., Fabregat, A., Foster, J. M., Griss, J., Alpi, E., Birim, M., Contell, J., O'Kelly, G., Schoenegger, A., Ovelleiro, D., Perez-Riverol, Y., Reisinger, F., Rios, D., Wang, R., Hermjakob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PRoteomics+IDEntifications+(PRIDE)+database+and+associated+tools:+status+in+2013.">The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.</a> Nucleic Acids Res. 41, 1063-1069 (2013).</li></ol>

作者宣称没有利益冲突。

<html>的关键步骤，从耳蜗感觉上皮最大化蛋白质鉴定是：1）使用多个endoproteinases的消化，2）使用多个分离技术，以及3）利用高分辨率质谱仪。多种酶的应用增加了肽的数目，提高了蛋白质序列覆盖率，从而提高识别的蛋白的数量从耳蜗组织。胰蛋白酶，最常用的蛋白酶提供蛋白质的效率和特异性裂解，产生的肽，是良好的MS电离和碎裂。然而，使用前胰蛋白酶另一种酶，如LYSC，这还能断裂在赖?...

蛋白质组学是复杂的生物系统，通过分析蛋白的表达，功能，修改和相互作用1的研究。几种方法已用于内耳，包括抗体微阵列2，二维凝胶电泳3-5，和DIGE 6的蛋白质组分析。然而，蛋白质的仅有限数量已被鉴定和表征2,7-10相比，在内耳11,12确定了超过10,000个基因和表达序列标签（ESTs），MS是最常用的和全面的技术蛋白质组学中蛋白质特征。复杂蛋白质样品，例如耳蜗的分析，是具有挑战性的。然而，多个分离技术与MS的结合，使更多数量的肽和蛋白质的鉴定，由于增加了动态范围的浓度和峰容量13。多维chromatograPHY通过允许使用不同的吸附机理降低高度复杂的蛋白质混合物。有两种常用的MS蛋白质组分析方法，猎枪和自下而上的蛋白质组学研究。在鸟枪蛋白质组学，完整蛋白质的混合物酶促消化，并且使用多维色谱用强阳离子交换色谱法（SCX），随后通过反相液相色谱法（RPLC）14,15分离。分离的肽进行串联质谱和数据库搜索15。这种技术的一个主要优点是，成千上万的蛋白质可以在一次分析中被识别和技术更适合于膜蛋白。 在自下而上的方法，所述蛋白质混合物分离，通常是由一维或二维电泳，并且单个蛋白质条带或斑点切出和消化酶如胰蛋白酶，通常会导致多种肽。然而，另一个更近LY开发的电泳方法，在自下而上的蛋白质组学使用，是GELFrEE。这种技术分馏蛋白质样品在液相，使它们较少分析前复杂。这种技术是可重复的，具有高的蛋白回收率，并降低高丰度蛋白的复杂蛋白样品16中的分布情况。肽，从分离的蛋白质产生的，是由MS分析，利用肽质量指纹或串联质谱（MS / MS），创建序列标签数据库搜索17-19。一些使用自底向上方法的主要优点是能够获得高分辨率的分离和蛋白质的全面覆盖的能力。自下而上蛋白质组学是蛋白质组学20的最广泛使用的技术，因此，一些生物信息学工具是可用的。此外，蛋白质可以被分离在消化之前的复杂混合物，所以鉴定的机会较大。 其中一个主要的挑战使用内耳的蛋白质组学分析是它的体积小，限制可访问性和细胞类型的多样性21。此外，区分它的功能的关键的蛋白质，如离子通道，转运蛋白和受体，是膜蛋白，它可以是难以分离22。因此，过滤器辅助样品制备（FASP）是有利的组织，这对于蛋白提取有限的蛋白质组学分析，并要求清洁剂来溶解膜23。该滤波可用于膜和可溶性蛋白的质谱分析和对从低分子量的污染物23,24分离的肽的能力。 本协议描述了常用的蛋白质组的方法，它们被组合和修改，以分析可溶性蛋白和膜蛋白，并从耳蜗感觉上皮蛋白最大化ID的数目。我们将介绍使用鸟枪蛋白质组学与FASP多消化离子，离子交换色谱法，高分辨质谱和数据分析。此外，我们将描述自下而上的蛋白质组学与GELFrEE，FASP多的消化，高分辨率质谱和数据分析。

<table border="0" cellpadding="0" cellspacing="0" width="749">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">8% Tris-acetate cartridge&#160;</td>
			<td style="width:159px;">Protein Discovery</td>
			<td style="width:133px;">42103</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Honeywell</td>
			<td>015-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AEBSF</td>
			<td>Calbiochem</td>
			<td>101500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>AC16861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Calbiochem</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ASB-14&#160;</td>
			<td>Calbiochem</td>
			<td>182750-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>BioRad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">C18 column&#160;</td>
			<td>New Objective</td>
			<td>A25112</td>
			<td>75 &#956;m &#215; 10 cm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DC Protein Assay</td>
			<td>BioRad</td>
			<td>500-0116</td>
			<td>Microplate Assay Protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endoproteinase Lys-C</td>
			<td>Sigma-Aldrich</td>
			<td>P3428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FASP Protein Digestion Kit</td>
			<td>Protein Discovery</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid&#160;</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GELFrEE Fractionation System</td>
			<td>Protein Discovery</td>
			<td>42001</td>
			<td>GELFrEE 8100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Calbiochem</td>
			<td>108975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MacroSpin Column</td>
			<td>The Nest Group</td>
			<td>SMM SS18V</td>
			<td>Silica C18</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcystin</td>
			<td>Calbiochem</td>
			<td>475815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysulfoethyl A Column</td>
			<td>The Nest Group</td>
			<td>202SE0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonic Dismembrator&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-338-53</td>
			<td>Model 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
			<td>Proteomics Grade</td>
		</tr>
	</tbody>
</table>

bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome

蛋白质组学是一种常用的方法，可以提供深入了解复杂的生物系统。耳蜗感觉上皮包含转导声音的机械能转换成由外周和中枢神经系统处理的电化学能量受体。几种蛋白质组技术已经发展到研究耳蜗内耳，如二维差异凝胶电泳（2D-DIGE），抗体微阵列，和质谱（MS）。 MS在蛋白质组学研究中最全面，最灵活的工具，并与分离方法结合使用可以提供一个深入蛋白质组生物样品。分离方法结合MS具有丰富的蛋白质样品，检测低分子量和疏水性蛋白质，并通过降低蛋白质的动态范围确定的低丰度蛋白的能力。不同消化策略可以应用到整个溶胞产物或分馏的蛋白裂解液来提高多肽和蛋白质序列覆盖率。不同的分离技术，包括强阳离子交换（SCX），反相（RP），和凝胶洗脱液体馏分包封电泳（GELFrEE）利用可应用于降低样品的复杂性之前MS分析蛋白质鉴定。

取得耳蜗感觉上皮的最全面的蛋白质组，快速组织解剖先于蛋白提取和样品制备要求。两种蛋白质组技术都可以使用，猎枪和自下而上蛋白质组学。以制备样品鸟枪蛋白质组学，FASP消化过程被用作如图1所示。该FASP方法允许浓度的蛋白质，除去洗涤剂，并用多种酶蛋白的消化。有两个双重消化过程中使用时，首先是胰蛋白酶消化接着进行第二用胰蛋白酶消化，这被汇集，分级分离的SC...

Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

耳蜗感觉上皮蛋白质组分析可能是由于其体积小和具有挑战性的，因为膜蛋白很难分离和鉴定。两膜和可溶性蛋白质可以通过组合多个的制备方法和分离技术随着高分辨率质谱来识别。

自下而上和猎枪蛋白质组学鉴定一个全面的蛋白质组耳蜗

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

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JoVE Journal

Biology

18.6K 次观看.  University of South Florida. 耳蜗感觉上皮蛋白质组分析可能是由于其体积小和具有挑战性的，因为膜蛋白很难分离和鉴定。两膜和可溶性蛋白质可以通过组合多个的制备方法和分离技术随着高分辨率质谱来识别。 

视频: Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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 ...

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 ...

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 ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

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

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

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.