作者根据批准号：0719232，美国国家科学基金会的美国能源部（DEFG02 01ER15129）对这项工作的财政支持表示感谢，和开普敦生物科学公司（摩根，西弗吉尼亚州）。作者还要感谢她的帮助下在议定书摄录杰西卡A. Stolee。

以下协议描述激光烧蚀电喷雾电离（LAESI）实验的主要步骤，并提供了横向和三维（3D）成像的动物和植物组织样本有代表性的例子。附加的实验和技术细节，可以从其他地方获得。 1-6 1。组织准备和安装<ol><li>如果需要切片，使用第一个cryomicrotome组织为10-100微米厚的切片在-10 -20 ° C，除非另有一个特定的组织类型的建议。 </li><li>装载到一个平坦的表面化学改性剂的情况下直接（例如，化学预清洁玻片上）的部分。切片组织，解冻的部分安装和安全的样品架，立即到佩尔蒂埃冷却阶段后解冻安装保留在任何时候都冻结在分析过程中的组织。这一步是需要尽量减少/防止分子中的部分迁移。 </li><li>如果需要，使用散热器配备了一个低功率的风扇，以方便散热佩尔蒂埃阶段保持组织冷冻。 </li><li>在潮湿的环境下超过延长期的时间（1-2小时），检查组织表面上凝结的水或冰。组织上的冷凝水产生不利影响LAESI实验成像性能。 </li><li>如果需要，使用一个房间除湿机或冷却样品放置在充满惰性气体（例如，干燥的氮气），以防止结露的环境室1 。 </li></ol> 2。 LAESI离子源的优化 LAESI离子源包括一个中红外激光，一系列光学元件光转向和重点，以及额外的样品架，散热元件，翻译阶段和溶剂输送系统。图1展示了这些元素的典型安排大气压离子源的质谱仪入口。 <ol><li>所示在图1位置质谱仪采样锥（D 或- FP）口下面的示例15-20毫米。 </li><li>操作在中红外激光波长2​​.94微米和10 Hz重复率。衰减〜100μJ/脉冲能量的激光输出。 </li><li>使用透明的激光波长（例如，一个平凸氟化钙或硒化锌透镜）金镜和聚焦透镜组合进样在正常发病率的激光脉冲能量（见右入射角与样品表面的情侣在图1）。 </li><li>质谱仪采样锥孔前中期红外光束轴5-8毫米的位置。 </li><li>调整对焦镜头位置和脉冲能量的激光束实现焦斑组织切除。消融量的尺寸决定了成像应用的像素（或三维成像体素）的大小。 </li><li>在质谱仪的入口轴和纳流喷雾发射器的距离（见图1）〜10毫米的孔到发射极的尖端位置。 </li><li>电，准备用50％甲醇溶液，分别用0.1％醋酸或0.1％醋酸铵积极或消极的离子模式添加剂。根据样品，等有机溶剂，如乙腈，异丙醇等，可能会取代在适当浓度的分析任务甲醇。电稳定是成功的成像的关键。根据溶剂的选择，流量和喷涂电压需要进行调整，以实现稳定的喷涂。 </li><li>成像应用，无功LAESI 6 electrosprayed解决方案可能包含反应物。 </li><li>使用注射泵通过电发射器提供一个〜300 NL / min的流速电的解决方案。 </li><li>如果质谱仪口保持在低电压（&lt;500 V的对地测量），通过施加高电压电发射器（例如，3000伏）直接或通过一个金属工会产生电。否则，地面直接电发射器或通过金属工会建立电。 </li><li> LAESI喷洒最有效的离子生成模式操作锥形喷射电源。对于操作变量喷洒模式和质谱其效果的影响，其他地方看到讨论 。5,7,8 </li><li>仔细的LAESI设置调整优化，同时保持在同一平面上的激光束，发射器，和孔轴LAESI离子产量的相对距离。请其他地方找到详细说明。 </li><li>用光学显微镜，确定消融火山口的横向尺寸的样品。 </li><li>立体LAESI成像实验，执行与个别脉冲的消融，并确定使用体素的深度，例如，在光学显微镜的Z - Stack的模式。 </li></ol> 3。分子成像和数据分析在成像实验，移动组织样本中的一步尺寸大于或等于消融点的尺寸的X和Y方向的激光焦平面。空间分辨率是有限的，由入射激光束聚焦。 <ol><li>选择在样品表面的兴趣领域，并取得相应的边界坐标（X，Y，）。 </li><li>选择一个网格算法（例如，自适应网格，选定区域成像，矩形网格，螺旋纹，Z轴扫描等）与光栅样品表面与选择住在每个像素在成像领域的时间。 </li><li>使用三轴​​的翻译阶段，软件，光栅样品，按照预定的电网。 </li><li>计算成像所需的总时间。 </li><li>禁用质谱仪数据采集的时间限制。如果这是不可能的，集数据采集，计算成像时间值的时间限制。 </li><li>开始在一个适当的重复率的中红外激光源产生足够的信号噪声比质谱，在每个像素内的停留时间，执行LAESI横向成像实验。三维分子成像技术，使用一个比激光源的重复率，成功地大规模分析在一个单一的激光脉冲产生的离子光谱采集率更高。等待START信号启动消融序列。 </li><li>开启的电源。确保有足够的解决方案是成像所需的全部时间。 </li><li>同时启动收购质谱，中红外激光烧蚀，表面扫描。 </li><li>当成像运行完成后，停止表面扫描，中红外激光，和数据采集。 </li><li>禁用激光源。 </li><li>关闭高电压。 </li><li>关闭注射泵。 </li><li>质谱仪设置到待机模式。 </li><li>关闭帕尔帖冷却电子。 </li><li>关闭如果使用惰性气体的气流。 </li><li>使用软件关联在外侧成像或体素的像素的三维分析中的绝对坐标与相应的光谱。 </li><li>为选定的m / z对获得横向和三维的分子图像分析的绝对坐标值绘制的离子强度信号。 </li></ol> 4。代表性的成果图2给出了一些主要的组织类型和成像代表性的结果。 A组描绘了一个已冻结在实验过程中，以防止脱水的动物组织切片的情况下，1此外，设在干燥的氮气环境样品，以避免周围的水蒸汽凝结在样品表面。一个100微米厚的大鼠脑冠状断面（ 褐家鼠 ）横向成像LAESI。光学图像的大脑解剖区域（见面板）显示的plasmalogens PC获得的分子图像（O - 33：3）良好的相关性和/或PE（O - 36：3）m / z为 728.559。 B组LAESI斑马的植物（Aphelandra子草 ）叶组织的三维成像。由于叶片具有自然防御机制防止脱水，样品可以在周围环境中的审问，3所获得的分子三维图像揭示了各种初级和次生代谢产物的分布格局。其中，金合欢素的m / z 285.076是从均匀分布在其他顶部的第二和第三层的黄色行业更高的离子计数检测。同意这种分配格局与光学图像中看到的花斑。 <img src="/files/ftp_upload/2097/2097fig1.jpg" alt="图1" /> 图1。 LAESI系统原理图（ES，电发射端，或质谱仪采样锥孔;佛罗里达州，聚焦镜头;计划生育，联络点，P，Peltier冷却阶段;房协，散热器） 。一个颗粒物的一部分，在中红外消融（红点）的电产生带电液滴与样品的分子和离子（绿色圆点）种子coalesces开除。从这些水滴释放的离子质谱仪分析和记录。 <img src="/files/ftp_upload/2097/2097fig2.jpg" alt="图2" /> 图2。横向和3D意马代表结果与吴LAESI质量法（ 一 ）顶部面板描绘老鼠大脑（ 褐家鼠 ）冠状切面的分子图像，为plasmalogens PC获得光学图像（O - 33：3）和/或PE（O - 36 ：3）m / z为 728.559。白色比例尺对应到1毫米。适应许可（参考文献1）。 2010年美国化学学会版权所有。 （二）底部面板上显示了一个从杂色斑马植物的叶（Aphelandra子草）的三维成像。 m / z为 285.076，刺槐素是从均匀分布在其他顶部的第二和第三层的黄色行业更高的离子计数检测。重印许可（参考文献3）。版权所有2009美国化学学会。

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	<li>Nemes, P., Woods, A. S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Imaging+of+Small+Metabolites+and+Lipids+in+Rat+Brain+Tissues+at+Atmospheric-Pressure+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric-Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Anal Chem. 82, 982-988 (2010).</li><li>Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure,+in+vivo,+and+imaging+mass+spectrometry.">Laser ablation electrospray ionization for atmospheric-pressure, in vivo, and imaging mass spectrometry.</a> Anal Chem. 79, 8098-8106 (2007).</li><li>Nemes, P., Barton, A. A., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+metabolites+in+tissues+under+ambient+conditions+by+laser+ablation+electrospray+ionization+mass+spectrometry.">Three-dimensional imaging of metabolites in tissues under ambient conditions by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 6668-6675 (2009).</li><li>Nemes, P., Barton, A. A., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+molecular+imaging+and+depth+profiling+of+live+tissue+by+infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 80, 4575-4582 (2008).</li><li>Nemes, P., Vertes, A., Nemes, S. S., Rubakhin, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+electrospray+ionization+for+atmospheric-pressure+molecular+imaging+mass+spectrometry.+Mass+Spectrometry+Imaging.">Laser ablation electrospray ionization for atmospheric-pressure molecular imaging mass spectrometry. Mass Spectrometry Imaging.</a> Methods in Molecular Biology. , (2010).</li><li>Shrestha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+lipids+and+small+metabolites+in+mouse+brain+tissue+by+AP+IR-MALDI+and+reactive+LAESI+mass+spectrometry.">Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.</a> Analyst. 135, 751-758 (2010).</li><li>Nemes, P., Marginean, I., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spraying+mode+effect+on+droplet+formation+and+ion+chemistry+in+electrosprays.">Spraying mode effect on droplet formation and ion chemistry in electrosprays.</a> Anal Chem. 79, 3105-3116 (2007).</li><li>Nemes, P., Goyal, S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+and+noncovalent+complexation+changes+in+proteins+during+electrospray+ionization.">Conformational and noncovalent complexation changes in proteins during electrospray ionization.</a> Anal Chem. 80, 387-395 (2008).</li><li>Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+by+Mid-IR+laser+ablation+mass+spectrometry.">Molecular imaging by Mid-IR laser ablation mass spectrometry.</a> Appl Phys A-Mater Sci Process. 93, 885-891 (2008).</li><li>Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+metabolic+profiling+of+single+cells+by+laser+ablation+electrospray+ionization+mass+spectrometry.">In situ metabolic profiling of single cells by laser ablation electrospray ionization mass spectrometry.</a> Anal Chem. 81, 8265-8271 (2009).</li><li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+mass+spectrometry.">Ambient mass spectrometry.</a> Science. 311, 1566-1570 (2006).</li></ol>

不同组织类型表现出不同的水含量和拉伸强度，这反过来，可能会影响样本的消融特点。9，以减轻这些影响，它是理想的激光能量，样品处理和分析的协议进行修订更改时主要的组织类型。 对于单细胞或更高的分辨率的调查，中红外光可耦合成一个削尖的，而不是一个聚焦透镜的光纤定位接近选定的单元格在一个组织内的纤维尖端10，LAESI分析，可以对一种单细胞的水平。 作为一个无标签环境电离质谱的离子源，11 LAESI组织中的生化过程的调查显示巨大潜力。 LAESI直接分析，横向和三维成像的增值收益，是一个新兴的生物分析工具，分析以及成像应用。

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<td>Mass spectrometer</td>
<td>&nbsp;</td>
<td>Waters Co. (Milford, MA)</td>
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<td>&nbsp;</td>
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<td>Mid-IR laser</td>
<td>&nbsp;</td>
<td>Opotek Inc. (Carlsbad, CA)</td>
<td>Vibrant IR</td>
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atmospheric-pressure molecular imaging of biological tissues and biofilms by laesi mass spectrometry

环境电离质谱方法允许直接组织或母语一样的实验条件下的生物膜进行分析调查。激光烧蚀电喷雾电离（LAESI）就是这样的发展，特别是水含标本调查非常适合。 LAESI采用了中红外激光束（波长为2.94微米），以激发样品中的水分子。的消融能量密度超过临界值时，样品材料中的颗粒物的形式被驱逐，这些弹丸旅行到几十毫米以上的样品表面。 LAESI，这消融羽被拦截高度带电液滴捕捉到弹出的样品材料的一小部分，并将其转换成气相离子化学成分。一个大气压离子源接口配备一个质谱仪分析和记录离子的释放，来自探测样品的面积（像素）组成。超过一个像素阵列系统的审讯打开分子成像探针分析模式的一种方式。 LAESI质谱成像的一个独特之处在于深入剖析，在与横向成像相结合，使三维（3D）分子成像。 〜100微米〜40微米，分别与当前的横向和深度决议，LAESI质谱成像有助于探索生物组织的分子结构。在这里，我们审查了LAESI系统的主要元素，并提供一个成功的成像实验的指导方针。

Watch this Scientific Journal Video about Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry at JoVE.com

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

激光烧蚀电喷雾电离（LAESI）大气压离子源质谱。在成像模式下，中红外激光探测跨组织切片或生物膜的分子分布。这种技术提供了一个原生的实验条件下进行的各种生物分析研究的新方法。

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

atmospheric-pressure-molecular-imaging-biological-tissues-biofilms

Research

JoVE Journal

Biology

14.1K 次观看.  George Washington University. 激光烧蚀电喷雾电离（LAESI）大气压离子源质谱。在成像模式下，中红外激光探测跨组织切片或生物膜的分子分布。这种技术提供了一个原生的实验条件下进行的各种生物分析研究的新方法。

视频: Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI 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

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用：根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

高尔基蛋白质荧光质量中心的定量定位

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...