This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.

1.转<ol><li>种子在100毫米培养皿（2×10 6个细胞/皿）HEK293细胞。培养在补充有10％胎牛血清，100μg/ ml的链霉素和100单位/ ml青霉素的细胞在Dulbecco改进的Eagle氏培养基。孵育在加湿培养箱将细胞在5％CO 2和37℃。 </li><li>第二天，转染细胞10微克FLAG标记ARIP4质粒按照制造商的指示11，用40微升转染试剂用。 </li></ol> 2.蛋白提取<ol><li>约36小时转染后，立即用冰冷的PBS将细胞并使用刮刀收获细胞。转移细胞到1.5 mltube并在4℃下以8000 xg离心离心它为2分钟。吸出上清，重悬细胞沉淀在5×0.4米氯化钾缓冲的沉淀量（高盐缓冲液:20毫摩尔的Tris-HCl（pH为8.0），0.4M氯化钾，5mM的MgCl 2的，10％甘油，0.1％NP-40，10毫B-巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。旋转该混合物在4℃下20分钟。 </li><li>离心管中，在17700 XG在4℃下10分钟。 </li><li>上清液（全细胞提取物）转移到新的2毫升管中，并添加3×0米氯化钾缓冲的初始体积（稀释缓冲液:20毫摩尔的Tris-HCl（pH值8.0），5mM的MgCl 2的，10％甘油， 0.1％的NP-40，10毫β巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。 </li><li>执行另一个离心（17700×g离心4℃10分钟），上清液（全细胞提取物）转移到新的2ml管中。 </li></ol> 3.免疫沉淀<ol><li>在离心（2.4），洗50微升用PBS抗FLAG珠，0.1M甘氨酸 - 盐酸盐，然后的1M Tris盐酸，然后洗涤两次，用0.1M氯化钾缓冲液（低盐缓冲液:20毫摩尔三 - 盐酸（pH值8.0），0.1M氯化钾，5mM的氯化镁，10％甘油，0.1％NP-40，10μMM B巯基乙醇，1×蛋白酶抑制剂鸡尾酒）。此洗涤过程中离心分离是在2000 xg离心在4℃下进行1分钟。 </li><li>混合50微升FLAG珠与全细胞提取物，并在2-4℃（从步骤2.4全细胞提取物的1％也应保持在-80℃以供将来使用轻轻旋转混合物4小时为输入）。 </li><li>样品转移到微离心柱（重力下降）。保持流通在1.5ml管中，利用液氮冷冻，然后将其存储在-80℃下（检查输入和用Western印迹分析的流通的蛋白水平）。 </li><li>加入1 ml 0.1米氯化钾缓冲液（低盐缓冲液）的洗FLAG珠（重力下降）。 </li><li>重复步骤3.4至少四次（对总共五个洗涤）。 </li></ol> 4.洗脱<ol><li>放置柱到1.5毫升管，并在2000 xg离心离心它为1分钟;珠会干。 </li><li>盖的底列。 </li><li>添加50微升（等于FLAG珠的体积）0.1M甘氨酸 - 盐酸（pH值2.5）的。 </li><li>轻轻晃动，在室温下10分钟的混合物。 </li><li>放置柱到一个新的1.5毫升管，并在2000 xg离心离心它为1分钟。 </li><li>洗脱溶液与10微升的1M的Tris-HCl（pH 8.0）中的中和;通过移液或者通过使用涡旋机拌匀。 </li></ol> 5.烷基化和胰蛋白酶消化<ol><li>添加100mM的NH 4 HCO 3，10微升CH 3 CN的，和2μl的二硫苏糖醇的混合物（112微升总体积）的50微升。 </li><li>在56℃孵育样品30分钟。 </li><li>放置在室温下的样品10分钟。 </li><li>添加10微升333毫摩尔的碘乙酰胺的样品，并在37°C孵育它在黑暗中30分钟。 </li><li>添加胰蛋白酶的10微升（33纳克/微升）到混合物中，并在37℃过夜孵育它。 </li></ol><ol><li>以下过夜胰蛋白酶，运到最终产品的LC-MS / MS设施或用于分析12第三方质谱实验室。 </li></ol>

<ol><li>De Las Rivas, J., Fontanillo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-protein+interactions+essentials%3A+key+concepts+to+building+and+analyzing+interactome+networks%5BTitle%5D)+AND+%22PLoS+Comput.Biol%22%5BJournal%5D)">Protein-protein interactions essentials: key concepts to building and analyzing interactome networks.</a> PLoS Comput.Biol. 6 (6), e1000807(2010).</li>
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<li>MacPherson, R. E., Ramos, S. V., Vandenboom, R., Roy, B. D., Peters, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+PLIN+proteins%2C+ATGL+and+CGI-58%2C+interactions+at+rest+and+following+stimulated+contraction%5BTitle%5D)+AND+%22Am.J.Physiol.Regul.Integr.Comp.Physiol%22%5BJournal%5D)">Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction.</a> Am.J.Physiol.Regul.Integr.Comp.Physiol. 304 (8), 644-650 (2013).</li>
<li>Qin, K., Dong, C., Wu, G., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactive-state+preassembly+of+G%28q%29-coupled+receptors+and+G%28q%29+heterotrimers%5BTitle%5D)+AND+%22Nat.Chem.Biol%22%5BJournal%5D)">Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers.</a> Nat.Chem.Biol. 7 (10), 740-747 (2011).</li>
<li>Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M., Nakatani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complex+with+chromatin+modifiers+that+occupies+E2F-+and+Myc-responsive+genes+in+G0+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.</a> Science. 296 (5570), 1132-1136 (2002).</li>
<li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinated+histone+modifications+mediated+by+a+CtBP+co-repressor+complex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Coordinated histone modifications mediated by a CtBP co-repressor complex.</a> Nature. 422 (6933), 735-738 (2003).</li>
<li>Huh, K. W., DeMasi, J., Ogawa, H., Nakatani, Y., Howley, P. M., Munger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+the+human+papillomavirus+type+16+E7+oncoprotein+with+the+600-kDa+retinoblastoma+protein-associated+factor%2C+p600%5BTitle%5D)+AND+%22Proc.Natl.Acad.Sci.U.S.A%22%5BJournal%5D)">Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600.</a> Proc.Natl.Acad.Sci.U.S.A. 102 (32), 11492-11497 (2005).</li>
<li>Huang, B. X., Kim, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effective+identification+of+Akt+interacting+proteins+by+two-step+chemical+crosslinking%2C+co-immunoprecipitation+and+mass+spectrometry%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry.</a> PLoS One. 8 (4), 61430(2013).</li>
<li>ten Have, S., Boulon, S., Ahmad, Y., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry-based+immuno-precipitation+proteomics+-+the+user%27s+guide%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Mass spectrometry-based immuno-precipitation proteomics - the user's guide.</a> Proteomics. 11 (6), 1153-1159 (2011).</li>
<li>Ogawa, H., Komatsu, T., Hiraoka, Y., Morohashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+Suppression+by+Transient+Recruitment+of+ARIP4+to+Sumoylated+nuclear+receptor+Ad4BP%2FSF-1%5BTitle%5D)+AND+%22Mol.Biol.Cell%22%5BJournal%5D)">Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated nuclear receptor Ad4BP/SF-1.</a> Mol.Biol.Cell. 20 (19), 4235-4245 (2009).</li>
<li>Tsuchiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+autophagic+receptor+p62+regulates+the+abundance+of+transcriptional+coregulator+ARIP4+during+nutrient+starvation%5BTitle%5D)+AND+%22Sci.Rep%22%5BJournal%5D)">Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation.</a> Sci.Rep. 5, 14498(2015).</li>
<li>Watanabe, T., Matsuo, I., Maruyama, J., Kitamoto, K., Ito, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+an+intracellular+lectin%2C+calnexin%2C+from+Aspergillus+oryzae+using+N-glycan-conjugated+beads%5BTitle%5D)+AND+%22Biosci.Biotechnol.Biochem%22%5BJournal%5D)">Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads.</a> Biosci.Biotechnol.Biochem. 71 (11), 2688-2696 (2007).</li>
<li>Sitz, J. H., Tigges, M., Baumgartel, K., Khaspekov, L. G., Lutz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dyrk1A+potentiates+steroid+hormone-induced+transcription+via+the+chromatin+remodeling+factor+Arip4%5BTitle%5D)+AND+%22Mol.Cell.Biol%22%5BJournal%5D)">Dyrk1A potentiates steroid hormone-induced transcription via the chromatin remodeling factor Arip4.</a> Mol.Cell.Biol. 24 (13), 5821-5834 (2004).</li>
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</ol>

The authors have nothing to disclose.

有效转染是必不可少的获得与该协议的成功的结果。因此，我们建议您使用Western blot分析，以确定免疫沉淀的FLAG标签的蛋白质水平。这个步骤允许用户以验证他们感兴趣的蛋白被正确过度表达，此外，该IP被成功执行。 FLAG标签的蛋白质水平也应之前的质谱分析进行检查。 有模拟样本应该被用作阴性对照，以避免得到假阳性结果，并以鉴别从真相互作用的背景。此外，研究人类蛋白质时，实验应在相对干净的环境（ 例如，洁净室）中进行，以避免污染。虽然具有高盐浓度（0.3-0.4 M）的蛋白质提取缓冲液可被偶尔用于提取核蛋白，具有0.1-0.15 1M盐的浓度的IP缓冲液是大多数十大推荐。这使得IP步骤而不破坏蛋白质 - 蛋白质相互作用进行。为了进一步减少背景信号，磁珠可以用于纯化过程。同样重要的是，优化了细胞裂解条件，以降低假阳性结果。 在这项研究中相对于其它技术所描述的方法的显着性（基于消化例如，在凝胶单一蛋白识别）5，10，11是它允许用户为高通量鉴定进行LC-MS / MS分析核辅助因子及其结合合作伙伴之间的蛋白相互作用。转录的基因调控是高度依赖于序列特异性DNA结合蛋白的合作。因此，转录因子通常具有大量的辅因子相互作用，以改变靶基因的表达。钍<html> erefore，以更好地理解这些管理机制转录，确定促进靶蛋白的活性是最重要的转录合作伙伴的细胞活动。我们的方法允许用户通过他们感兴趣的转录因子快速识别假定核辅助因子，并进一步加强了我们的转录调控机制的认识。 FLAG肽也可被用来代替0.1M甘氨酸 - 盐酸溶液（pH 2.5）中洗脱过程。然而，如果采用此变形例中，用于稀释FLAG肽的缓冲液的pH应被监控。 3X FLAG肽也应该用于从FLAG珠洗脱蛋白。因此，具有相对高的pH值的溶液，应使用以稀释这些修饰的条件下，FLAG肽。它也至关重要，以避免以保持适当的蛋白质 - 蛋白质相互作用的样品的反复冻融。 t"的&gt;有时，胰蛋白酶消化过程的优化，可能还需要用于提高蛋白质相互作用筛选结果的准确性。虽然在LC-MS / MS法是可以用来识别核之间的相互作用的工具蛋白质，它的局限性之一是，它不提供关于该蛋白质复合物的装配准确的信息。为了提高复合体稳定性，化学交联处理可以使用14。作为另一种方法，毛细管电泳-质谱/质谱也是有用的15 。

蛋白质 - 蛋白质相互作用发挥多种生物学功能中起重要作用。正因为如此，这些相互作用已牵涉信号转导;跨细胞膜蛋白质运输;细胞的新陈代谢;和几个核过程，包括DNA复制，DNA损伤修复，重组和转录1，2，3，4。查明参与这些相互作用蛋白质因此，推进我们的这些细胞过程的理解是至关重要的。 免疫沉淀（IP）是用于分析蛋白质 - 蛋白质相互作用经过验证的技术。为了便于共同免疫沉淀的蛋白的鉴定，质谱常常利用5，6，7，8，9。通过用抗体靶向的蛋白复合物的公知的部件，它是可以分离的蛋白复合物，并随后经由质谱分析确定其未知组分。 ARIP4（雄激素受体相互作用蛋白4），一个转录coregulator，核受体蛋白以激活或在一个依赖于上下文的方式9,10抑制其靶启动子相互作用。为了更好地了解控制这些核因子的转录调控机制，我们描述了一个全面的方法来净化和使用LC-MS / MS系统识别ARIP4相互作用的蛋白质。

<table><tbody><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (EDTA free) (100x)</td><td>nacalai tesque</td><td>03969-21</td><td></td></tr><tr><td>ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>Micro Bio-Spin Columns</td><td>BIO-RAD</td><td>732-6204</td><td></td></tr></tbody></table>

a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

我们确定了一个强烈的信号ARIP4，约160千道尔顿，以及那些一个模拟采样控制等几个未知的蛋白质（ 图1）。 LC-MS / MS分析，鉴定两ARIP4复杂肽和FLAG珠（ 表1）的级分中的潜在的肽辅因子。 P62（Sequestosome1），公知的辅因子ARIP4 11中的分析（ 表1），这证实了本系统11的功效被确定。我们还确定了与ARIP4互动其它许多以往报道的蛋白质。以这种方式，SUMO2 10和DryK 13经由LC-MS / MS分析进行检测。 ARIP4肽使用LC-MS / MS分析，这表明该IP是高品质也确定。总体而言，LC-MS / MS技术是可以用于识别未知核事件链接的强大工具编到蛋白质 - 蛋白质相互作用。全面质量分析结果将根据要求提供。 <img alt="图1" src="/files/ftp_upload/55077/55077fig1.jpg" /> 图1:ARIP4配合物的净化。 FLAG表位标记的ARIP4的银染（ARIP4-F），在HEK293细胞和免疫沉淀表达与特定FLAG抗体。纯化的蛋白用凝胶电泳分离并用银染色显示。作为对照，对HEK293细胞不表达外源蛋白质，进行一个模拟纯化。分子量标准显示在左侧。 <a href="http://ecsource.jove.com/files/ftp_upload/55077/55077fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/55077/55077table1.jpg" /> 表1:辨识新型ARIP4结合蛋白氮。结合到纯化的蛋白质复合物FLAG珠通过基于胰蛋白酶消化解离。用LC-MS / MS分析这些蛋白质随后鉴定。使用SWISS-PROT数据库和MASCOT软件在LC-MS / MS数据进行分析。肽的数量和这些蛋白质的身份被示出。只选择与显著MASCOT评分（P &lt;0.05）的蛋白质。

Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

我们已经建立了使用LC-MS / MS系统共同调节相互作用蛋白的纯化的方法。

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

a-protein-preparation-method-for-high-throughput-identification

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.3K 次观看.  Osaka University. 我们已经建立了使用LC-MS / MS系统共同调节相互作用蛋白的纯化的方法。 

视频: A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

科研

生物化学

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

基于靶向质谱的低丰度蛋白质蛋白分析中的微量采样

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

化学

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

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

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

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

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

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

生物学

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

神经科学

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

摘要

探索更多视频

RNA 结合区质量谱鉴定的样品制备

使用膜内消化技术评估蛋白质与蛋白质相互作用

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

基于靶向质谱的低丰度蛋白质蛋白分析中的微量采样

分析甲基化和其他 S腺苷_{L同型半胱氨酸结合蛋白}

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

多蛋白复合物的比较分析新方法基于

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

A蛋白制备方法在蛋白质与核余子交互使用LC-MS / MS分析的高通量鉴定

摘要

探索更多视频

RNA 结合区质量谱鉴定的样品制备

使用膜内消化技术评估蛋白质与蛋白质相互作用

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

基于靶向质谱的低丰度蛋白质蛋白分析中的微量采样

分析甲基化和其他 S腺苷 L同型半胱氨酸结合蛋白

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定

多蛋白复合物的比较分析新方法基于

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

具有邻近标记蛋白质组学的神经元溶酶体相互作用组表征

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

分析甲基化和其他 S腺苷_{L同型半胱氨酸结合蛋白}