这项工作是资助由美国国立癌症研究所临床蛋白质组学的技术评估中心（CPTAC）授予（＃U24 CA126476）以及授予的乳腺肿瘤生物标志物发现联盟的娱乐产业基金会（EIF），并在EIF的妇女的癌症研究基金，并凯克基金会，加那利基金会，和保罗G. Allen的家庭基金会的厚礼。

实验过程： 该试剂盒需要合成肽和抗肽抗体。应该是唯一的选择肽感兴趣的蛋白质，含有8至22种氨基酸，并没有已知的翻译后修饰。蛋氨酸残留一般都避免和肽含有二元氨基酸（如KK，韩国KR，RR），都是不可取的。这种技术，它是共同使用内部标准的稳定同位素标记肽，结合重（13 C 和15 N）在C -末端肽（即K或R标记）标记的氨基酸。 以下协议描述来衡量鼠标蛋白骨桥蛋白肽GDSLAYGLR，使用发邮件至公司（伯林格姆，CA）和新英格兰肽（加德纳，马）的合成肽获得的抗肽抗体检测。该协议包括三个主要步骤（图1）：1）胰酶消化复杂的蛋白质混合物，2）肽的富集3）质谱分析。这将被证明添加用鼠标骨桥蛋白在人体血浆样品。 1。胰蛋白酶的酶消化和清理<ol><li>湿冰解冻10μL整齐的血浆等份。 </li><li>确定由BCA法测定总蛋白浓度和离心机样本，以消除任何悬浮固体。 </li><li>吸取10μL分装，从它的存储管1000μL深孔板和皮尔斯，能够薄膜盖。 </li><li>每个样品中加入20μL新鲜9M尿素/ 30MM二硫苏糖醇（DTT）（终浓度为6M尿素/ 20mm的数码地面电视）。 </li><li> 30分钟在37 ° C。 </li><li>每个样品中加入3μL新鲜的500毫米碘乙酰胺（50MM最终的IAM）。 </li><li>在室温下孵育30分钟，在黑暗中。 </li><li>加入257μL的100毫米Tris（pH值8）（稀释尿素〜0.6M）。 </li><li>添加10μL胰蛋白酶原液（1微克/微升; 1:50酶底物比例）。 </li><li>孵育37℃过夜（12-16小时）。 </li><li>加入3μL整齐的甲酸（终浓度为1％）。 </li><li>添加稳定同位素标准（如果执行一个复用的检测，通常约10μL，含有50-100 fmol的标准同位素标记的肽是多个标准添加）。 </li><li>绿洲盒板洗净，与500μL0.1％甲酸80％乙腈，丢弃流量通过。重复此3倍。 </li><li>加入500μL0.1％甲酸水平衡墨盒板，并丢弃的流过。重复此的4倍。 </li><li>消化样品装入墨盒板和调整的真空，所以流动是非常缓慢。 </li><li> 500μL0.1％甲酸水清洗，并丢弃流过。重复此3倍。 </li><li> 2 × 500μL0.1％甲酸加入到1000μL深孔板的80％乙腈洗脱肽（不要丢弃流过）。 </li><li>冻干（或speedvac）洗脱液干燥。 （冻干是首选的方法） </li><li>重组干肽，加入50μL的PBS + 0.03％CHAPS。 </li></ol> 2。肽免疫浓缩<ol><li>将样品标准翠鸟96孔板。 </li><li>新增1微克抗体和1.5μL蛋白- G的磁性珠每个目标（交联抗体珠之前，除了它是可选的）。确保珠以及暂停摇晃或震荡。 </li><li>盖的板带箔。 </li><li>孵育过夜（12-16小时），轻轻翻滚，以确保珠被暂停。 </li><li>离心5秒32 XG板箔表面除去任何液体​​。 </li><li>拆下的铝箔盖。 </li><li>洗涤和洗脱珠可以进行手动或自动方式。此过程介绍了翠鸟平台上自动化的步骤。 </li><li>洗磁珠200μL的PBS + 0.03％CHAPS（每洗1分钟）的2倍。 </li><li>洗磁珠200μL1:10稀释的PBS + 0.03％CHAPS（1分钟）1次。 </li><li>肽洗脱在25 5％醋酸+ 0.03％CHAPS UL。 </li><li>将一块磁铁洗脱板和洗脱液转移到96孔板中，注意不要转移剩余的珠子。 </li><li>盖密封垫盘。 </li><li>板包含洗脱液交付的三倍quadrapole分析质谱仪。 </li></ol> 3。多反应监测分析 - 质谱<ol><li> SRM / MRM分析的转换，可以选择和优化，通过注入0.5μL/ min的质谱仪1 picomole每微升肽标准的解决方案在30％acetonitrile/0.1％甲酸。一旦获得稳定的喷涂，收集的MS / MS谱。 </li><li>转换选择的MS / MS谱确定THRE个人在噪音小的频谱地区丰富的碎片离子。对于乘电荷的多肽离子，检测Y -离子碎片为m / z的m / z&gt;易制毒化学通常是最好SRM / MRM检测发展的。图2显示的MS / MS谱和选定的过渡离子476.3&gt; 508.3 ，579.3肽GDSLAYGLR 692.4（转变为重稳定同位素标准481.3&gt; 518.3，589.3，702.4未显示）。 </li><li>根据制作和使用分光计的质量模型，也有多个参数，可以为每个过渡优化。在这里，我们通过提高碰撞能量和监测的信号电平优化每个选定的过渡的碰撞能量。一个25的碰撞能量被用于每个过渡。 </li><li>简单来说，是一个典型的配置SRM / MRM分析如下：流动相（A）0.1％的甲酸;（二）90％的乙腈/ 0.1％甲酸，0.3 × 5毫米C18陷阱列，编号75微米× 15厘米C18分析柱（Reprosil - PUR C18 AQ，120 °孔）。注射量为10μL，样品装载在总流量在3％B级为5分钟，3μL/ min的线性梯度洗脱从3 - 45 300％B级 - 400 NL /分钟，10分钟内。在4000 QTRAP（ABSCIEX，福斯特城，加利福尼亚州）的条件，喷雾电压2.3kV，离子源温度150℃，12日，GS1的窗帘气15。 </li><li>样品分析SRM / MRM注入10μL的样品。峰面积集成14计算无标签/标记在每个样本最丰富的过渡，是免费的背景噪音，在这种情况下，使用肽的峰面积比（PAR），Y5过渡使用的程序天际线的轻型和重型肽。 （光肽，重标准肽481.3&gt; 589.3 476.3&gt; 579.3）。 </li></ol> 4。代表性的成果： 测得的峰面积比值（轻内源性肽相对飙升的重同位素标记的肽）提供的目标肽的定量测量。图3显示了在一个SISCAPA丰富的样品的轻型和重型肽例如色谱。请注意，轻型和重型肽洗脱同时和多个过渡可用于确认身份的每个肽监测。 <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="图1"/> 图1。SISCAPA过程的示意图。是一个复杂的蛋白质混合物消化成肽。丰富蛋白质的G -磁性粒子固定化反肽抗体针对性肽分析物（内源性分析物和一个飙升的稳定同位素标记的内部标准）。以下隔离，有针对性的肽洗脱的磁性粒子和质量法相对的内部标准定量分析。 <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="图2"/> 图2的MS / MS谱显示SRM / MRM转变的三个片段的肽GDSLAYGLR 。 <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="图3"/> 图3。范例色谱显示轻肽待测峰形（红色）和沉重的稳定同位素标记的内部标准（蓝色） 。 Y5光肽（476.3&gt; 579.3）和沉重的稳定同位素标记的过渡色谱标准（481.3&gt; 589.3）随着时间的推移，他们绘制洗脱色谱系统。

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没有利益冲突的声明。

所述的协议中最关键的一步是确保珠保持在发病潜伏期间，充分混合。允许珠定居以及/瓶的底部，会导致变异增加。同样重要的是降速以及/瓶顶端的潜伏期后，可能会保留在任何液体。重复的胰蛋白酶消化也很关键。这里描述的消化过程已被广泛使用在我们的实验室与SISCAPA一起，但是，它是消化可能的替代方法，可以为一个给定的目标蛋白进行了优化的15游离抗体洗脱不干预的检测。肽，可能因为排除列或流出最迟肽抗体，但抗体可交联G蛋白珠之前，在大量的实验，或者如果游离抗体的亲和力浓缩成了一个问题。我们还发现它有用的地方一个自动进样器的样品板以及洗衣机在反向流动陷阱列下面的磁铁（加载），以除去残留的珠子或颗粒。除了 ​​磁珠描述的方法，这项技术也可以适应一个列的格式（即亲和层析），12， 16 一旦用户的总体协议感到满意，该技术也适合进行若干修改，提高整体检测。11首先，它有可能在浓缩步骤（即复）相结合的抗体，在一个单一的检测分析多种分析。质谱仪能够同时分析大量的分析物。其次，越来越多的原始样本量，提高了检测的灵敏度。

<table border="1"><tbody><tr><th>材料名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 1000ul深井96孔板</td><td> Eppendorf公司</td><td> 951032786 </td></tr><tr><td>绿洲HLB值1毫升墨盒板，9月白40毫克，96孔板</td><td>沃特斯</td><td> 186003966 </td></tr><tr><td>翠鸟96孔板</td><td>赛默飞世尔科技</td><td> 97002540 </td></tr><tr><td>清除96孔，白色的墙壁板</td><td> Bio - Rad公司</td><td> HSP9601 </td></tr><tr><td>箔96孔板盖板</td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Axymat 96孔板的密封垫</td><td>爱思进</td><td> 521-01-151 </td></tr><tr><td> X皮尔斯电影</td><td> Sigma - Aldrich公司</td><td> Z722502 </td></tr><tr><td>翠鸟磁珠处理器，用PCR磁铁头</td><td>赛默飞世尔科技</td><td>热休克蛋白9601 </td></tr></tbody></table>表1：材料<table border="1"><tbody><tr><th>材料名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>尿素</td><td>西格玛爱秩序</td><td> U6031 </td><td></td></tr><tr><td> Trizma基地</td><td>西格玛爱秩序</td><td> T1503 </td><td></td></tr><tr><td>二硫苏糖醇（DTT） </td><td>刺穿</td><td> 20291 </td><td> “无权衡二硫苏糖醇” </td></tr><tr><td>碘乙酰胺（IAM） </td><td>西格玛爱秩序</td><td> I1149 </td><td></td></tr><tr><td>胰蛋白酶黄金</td><td> Promega公司</td><td> V5280 </td><td></td></tr><tr><td> G蛋白的磁珠，2.8um </td><td> Invitrogen公司</td><td> 10004D </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td>赛默飞世尔科技</td><td> L5401 </td><td></td></tr><tr><td> CHAPS </td><td>赛默飞世尔科技</td><td> 28300 </td><td></td></tr><tr><td>甲酸</td><td>电解二氧化锰</td><td> 11670 </td><td></td></tr><tr><td>乙腈</td><td>赛默飞世尔科技</td><td> A998 - 1 </td><td></td></tr><tr><td>醋酸</td><td>西格玛爱秩序</td><td> 242853 </td><td></td></tr></tbody></table>表2：试剂<table border="1"><tbody><tr><th>解决方案</th><th>评论（可选） </th></tr><tr><td> 100毫米的Tris pH值8 </td><td></td></tr><tr><td> 9 M尿素30毫米/ 100毫米的Tris（pH值8）数码地面电视</td><td>必须准备新鲜每次</td></tr><tr><td> 500毫米的IAM在100毫米的Tris（pH值8） </td><td>必须准备新鲜每次</td></tr><tr><td>胰蛋白酶1mg/ml的100毫米的Tris pH值8 </td><td></td></tr><tr><td>胰蛋白酶抑制剂1mg/ml的100毫米的Tris pH值8 </td><td></td></tr><tr><td>稳定同位素标准mastermix </td><td></td></tr><tr><td>抗肽抗体mastermix </td><td></td></tr></tbody></table>表3：要准备的解决方案

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

定量分析测量蛋白质有一个很大的需要。与传统的夹心免疫，主要是考虑在定量的金标准，是一个成本高，很长的时间，和充满弊端（如异嗜性抗体，抗体的干扰，“勾效应”），1另一项技术是亲和富集再加定量质谱肽，俗称为SISCAPA（稳定同位素标准和反肽抗体捕获）2。这项技术，亲和富集稳定同位素稀释和选择/多反应监测质谱检测肽（ SRM / MRM - MS）作为代理人为各自的蛋白质肽的定量测量。 SRM / MRM - MS是公认的准确定量的小分子3，4，最近已经适应在血浆和细胞裂解液中蛋白质的含量来衡量。5-7实现定量的蛋白质，这些较大的分子被消化组件肽酶如胰蛋白酶。一个或多个选定的肽，其序列是唯一的在该物种的靶蛋白（即“proteotypic”肽），然后丰富使用抗肽抗体的样品，并测量样品中的蛋白质浓度的定量计量代理人。因此，加上稳定的同位素稀释（SID）的方法（即飙升的稳定同位素标记肽标准），SRM / MRM可以用来衡量作为代理人在复杂的生物基质蛋白定量proteotypic肽的浓度。这些化验有几个优点相比传统的免疫。浓缩试剂是相对较便宜的生成，分析物的特异性良好，检测可高度复用，可以进行从整齐的等离子（没有枯竭），和该技术是适合广泛的蛋白质或修改我们在这个视频的兴趣。8-13演示了基本的协议，适应磁珠平台。

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

稳定同位素标准和捕捉反肽抗体（SISCAPA）夫妇亲和富集稳定同位素稀释质谱（MRM - MS）的肽作为代理人为各自的蛋白质肽的定量测量提供。在这里，我们描述了一个部分自动化的格式使用磁性粒子的协议。

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 ...

quantification-proteins-using-peptide-immunoaffinity-enrichment

Research

JoVE Journal

Biology

22.7K 次观看.  Fred Hutchinson Cancer Research Center - FHCRC. 稳定同位素标准和捕捉反肽抗体（SISCAPA）夫妇亲和富集稳定同位素稀释质谱（MRM - MS）的肽作为代理人为各自的蛋白质肽的定量测量提供。在这里，我们描述了一个部分自动化的格式使用磁性粒子的协议。

视频: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

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

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 ...

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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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 ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

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

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

利用同位素标记的质谱剖析巯基氧化还原蛋白质

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

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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.

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 ...

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 ...