作者承认由以下机构的财政支持;，美国国家科学基金会（格兰特0719232），能源部（批准DEFG02 - 01ER15129），WM凯克基金会（格兰特041904），和乔治华盛顿大学的研究加强基金。作者感谢慷慨红外纤维系统（银泉，MD），以及提供初步的协议，讨论关于纤维由Mark E. Reeves和“乔治华盛顿”琼A.霍夫曼蚀刻二氧化锗基于光纤大学。作者还要感谢她的帮助下在议定书摄录杰西卡A. Stolee。

1。光学元件<ol><li>激光烧蚀激光烧蚀电喷雾电离（LAESI）质谱（MS）是由5 ns的激光脉冲的波长为2.94微米。一个可调谐光参量振荡器驱动的Nd：YAG激光（100 Opolette Opotek公司，卡尔斯巴德，CA）是用于在这项研究中消融。选择的重复速率是5和100赫兹之间。这是一个IV级激光直接照射后，可能会导致严重的永久性的损害眼睛或皮肤。漫反射的激光束也可以到皮肤或眼睛有害。穿戴合适的激光护目镜保护。 </li><li>二氧化锗的玻璃制成的光纤红外光纤系统公司（银泉，MD）获得。如果有HYTREL和聚酰亚胺纤维上的涂料按照下列指示以消除他们从两端。热1 - 甲基-2 - 吡咯烷酮至130-150 ° C的通风柜。沾纤维结束时，你想将这种液体地带大约1分钟或直到涂层开始软化和剥离。软化涂层浸入甲醇或异丙醇一分钟，并用不起毛的免费餐巾纸擦去任何剩余的涂料。除去涂层后，切割的蓝宝石刀片（KITCO光纤，弗吉尼亚海滩，弗吉尼亚州）的得分和轻轻贴紧光纤两端。 </li><li>为了实现所需的清晰度，化学蚀刻的光纤尖端的一端由1％（V / V）硝酸（试剂级）解决方案。垂直浸入纤维成溶液，初次接触后的300至500微米的深度选定结束。蚀刻约15分钟后，提示会自发地脱离酸表面。这与〜15微米的曲率半径削尖的纤维尖端成果。与去离子水冲洗，以消除任何酸残基。 </li><li>山在显微纤维（MN - 151，Narishige，日本东京）的蚀刻结束，并把它邻近的细胞表面高效的能量沉积（〜20微米）。 </li><li>举行的光纤裸光纤夹头（BFC300，锡斯基尤公司，资助传球，或）非蚀刻结束。光学对准，光纤夹头装在一个五轴翻译（BFT - 5，锡斯基尤公司，通行证，或资助）。耦合进光纤的重点由平凸氟化钙或到非蚀刻结束防反射涂层的ZnSe镜头（红外光学产品，明代，NY）束激光能量（0.5至1兆焦耳）。受保护的金镜（PF10 - 03 - M01，Thorlabs，牛顿，新泽西州）是用于引导到中红外激光束的聚焦镜头。 </li></ol> 2。电安装<ol><li>电系统，可使用以下几部分组成：金属导电全氟套圈，配件，油管套，针端口（如U - 435，M215，F - 331Nx F - 242X，9013，分别IDEX健康与科学的结合，构造橡树港，WA）以及熔融石英管（例如，CT360 - 100 - 50 - 5，新的目标公司，沃本，MA）。建议的发射器是由不锈钢制成，并有一个50微米的ID（例如，MT320 - 50 - 5 - 5，新的目标公司，沃本，马萨诸塞州）的锥形尖。 </li><li>准备50％甲醇溶液与0.1％的乙酸（V / V）的电。解决的办法是在200至300 NL /分钟注射泵（哈佛器械，Holliston，MA）的速度泵使用，例如，一个500μL注射器（81222，汉密尔顿公司，里诺，内华达州）。 </li><li>稳压电源（PS350，斯坦福研究系统公司，桑尼维尔，加利福尼亚），产生一个稳定的电2,800 3,000 V之间的高电压应用的金属工会。确保所有电气连接是安全的和正确的接地与屏蔽屏蔽。与暴露的高压直接接触可能会导致触电。 </li></ol> 3。样品处理<ol><li>从适当的来源获取活组织或细胞样本，并让他们像以前那样分析建议。此演示洋葱灯泡是从当地的商店购买。由外科手术刀纵向切球。一个规模数厘米长的段，分段成条状。一个完整的单层表皮组织的内在揭下。 </li><li>湿表面的表皮是用于安装预清洗玻璃显微镜玻片上的组织。由通用板的持有人装上定位的三轴翻译阶段（FP01，Thorlabs公司，牛顿，新泽西州）举行的幻灯片。收购后立即安装，以防止细胞退化的样品的质谱。 </li></ol> 4。可视化系统<ol><li>两个长途显微镜是用来选择的细胞进行分析，并保持削尖纤维尖端与细胞表面之间的距离。后者安装在15 ° -30 °，从表面测量角度。该系统组成的人翁远程视频显微镜5倍至无限远（7X变焦精密光学，光学埃德蒙，巴林顿，新泽西州）纠正与CCD摄像头（F131马林，盟军视觉技术装备的物镜（2平方米APO 5倍，三丰公司，神奈川县，日本） Stadtroda，德国）为监测和图像采集。 </li><li>第二个视频显微镜安装在样品表面成直角，从顶部的可视化细胞。本系统提供可视化反馈，超过消融和分析选定的单元格对齐纤维尖端。它包括一个7倍变焦精密光学（埃德蒙光学，巴林顿，新泽西州）装有10倍无穷大，纠正长工作距离物镜（2平方米APO 10倍，三丰公司，神奈川县，日本）和CCD相机。 </li></ol> 5。收购质谱<ol><li>打开激光，电和质谱仪，例如，一个四极杆飞行时间系统（Q - TOF总理，沃特斯，米尔福德，马）。 LAESI离子源的几何结构优化调整消融点和彼此之间的相对电发射器和质谱仪的孔口，以达到最大的离子强度的位置。 </li><li>选择使用的顶视图可视化系统的细胞进行分析的兴趣。选择后，将在样品台上，带来的直接低于光纤削尖的提示选定的单元格。使用侧视镜调整尖〜20微米的细胞表面的距离。 </li><li>在选定的单元格通过蚀刻纤维尖端发射激光脉冲。这一结果在细胞壁和细胞质中的颗粒物的形式弹射穿孔。 postionization后由电，产生的离子质谱仪及质谱收集记录。 </li><li>数据采集​​后，把激光，电和在待机状态下的质谱仪，或将其关闭。保持的消融A。 CEPA为光镜可选观察组织样本。 </li></ol> 6。代表性的成果消融成功的一个单细胞在细胞壁和收集了LAESI质谱爆破结果。通常一个不成功的实验结果没有消融和/或没有质谱。出于演示的目的，我们目前的结果，从一个单一的嵌入式一个答：表皮细胞LAESI - MS分析CEPA灯泡。图2a显示了一个代表一个细胞的采样后的光学显微镜图像。细胞壁是穿孔和穿孔的程度是有限与邻近细胞的界限。相邻的细胞似乎是完整的。图2b显示了从细胞产生一个代表LAESI质谱。多种检测到的离子的A. CEPA细胞，并基于对他们的准确群众，同位素分布格局，并在某些情况下，串联质谱，他们试探性地分配到内源性代谢物。从单个细胞中检测到的离子类似的观察分析，在相同的组织由多个单元格的传统LAESI - MS的13。 <img src="/files/ftp_upload/2144/2144fig1.jpg" alt="图1" /> 图1。 LAESI - MS的单细胞分析示意图。一个中红外激光脉冲耦合到光纤和交付置于玻片上的样本。削尖的纤维尖端光聚焦到靶细胞和能量沉积阵阵细胞。生产消融羽（红点）合并电柱（黑点）的水滴凝聚样品中特定离子生产（绿色圆点）。这些离子质谱仪（MS）分析和检测。两个长距离视频显微镜，显微镜1和显微镜2，利用监测的光纤尖端与细胞表面之间的距离，并选择一个细胞，分别进行分析。 <img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="图2a" /> 图2a。消融的标志，一个答一个单一的表皮细胞CEPA灯泡。相邻的细胞没有表现出明显的损坏，随后可单独分析。 <img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="图2b" /> 图2b。质谱对应一个单一的A。消融CEPA表皮细胞显示，小学和次生代谢产物的存在。

<ol>
	<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><li>Chen, Z. 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=Early+plume+expansion+in+atmospheric+pressure+midinfrared+laser+ablation+of+water-rich+targets.">Early plume expansion in atmospheric pressure midinfrared laser ablation of water-rich targets.</a> Physical Review E. 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细胞的细胞质和细胞壁或膜的拉伸强度的含水量都消融在LAESI - MS分析单细胞的关键因素。激光能量密度进行调整，以达到不同的细胞类型的消融阈值。交付的激光能量密度取决于激光脉冲的能量和纤维尖端与细胞表面之间的距离。尽量减少扰动邻近的细胞，在单细胞分析是至关重要的。烧蚀阈值保持在激光能量限制在相邻细胞的分析效果。 潜在的应用之一就是使用自然容积化学成像像素（体素）由MS的单细胞。人工往往是矩形网格上收集数据相比，一个组织一个细胞化学成像时间更可能保留空间组织的细胞间的生化相互作用。

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<td>Acetic acid</td>
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direct analysis of single cells by mass spectrometry at atmospheric pressure

在单个细胞的生化物质的分析是理解细胞代谢，细胞周期，适应，疾病状态等的重要，即使是相同的细胞类型表现出异质性的生化妆，根据他们的生理条件和与环境的相互作用。常规质谱（MS）用于在单细胞生物分子分析的方法依赖于广泛的样品制备。卸下从他们的自然环境和丰富的来样加工细胞可能会导致细胞成分的变化。环境电离方法使在其原生环境，并没有广泛的样品制备样品的分析，中红外（中红外）激光波长为2.94微米的生物材料的烧蚀技术利用的水突然激发，结果在第一阶段爆炸2环境中红外激光辐射，如激光烧蚀电喷雾电离（LAESI）和红外大气压力基质辅助激光解吸电离（AP红外MALDI），电离技术，成功地展示了能力直接分析水丰富的组织和biofluids在大气压力。LAESI，主要是中性的颗粒物质从高电荷的电液滴产生离子的样品coalesces组成的中红外激光烧蚀羽3-11。近日，中红外单细胞消融提供通过蚀刻光纤中红外辐射。从这个消融产生的羽postionized电能够在单细胞LAESI - MS分析多样化的代谢产物。本文介绍了单细胞LAESI - MS分析的详细协议。视频演示一个单一的洋葱灯泡皮肤表皮细胞的分析。该系统的原理图如图1所示。图2提供了一个有代表性的例子是单细胞消融和细胞LAESI质谱。

Watch this Scientific Journal Video about Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure at JoVE.com

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

使用锋利的光纤样品激光烧蚀电喷雾电离（LAESI）质谱细胞，单细胞分析是通过质谱在大气压力下的植物和动物细胞。

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 ...

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15.9K 次观看.  George Washington University. 使用锋利的光纤样品激光烧蚀电喷雾电离（LAESI）质谱细胞，单细胞分析是通过质谱在大气压力下的植物和动物细胞。

视频: Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

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. ...

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

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

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 ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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 ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

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 ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...