支持这项工作是由国家卫生部批准GM - 26738研究院（翟）的一部分。

1。 PAK2激活<ol><li>添加适当的预测试量的caspase 3至20μL缓冲液中12毫克/毫升PAK2（50毫米的Tris - HCl，150 mM氯化钠，2 mM的数码地面电视在pH 7.5）和34℃孵育30分钟完全切割和激活PAK2。 </li><li>分析裂解产物经SDS - PAGE（10％）和Coommassie蓝染色。 </li><li>添加裂PAK2激活缓冲液B（50毫米的Tris - HCl，150毫米15毫米氯化钠，氯化镁2，10毫米ATP）在pH 7.5，在20μL的终浓度为10毫克/毫升PAK2，并孵育34 ° C下30分钟，充分激活自身磷酸化在8个地点PAK2。 </li><li>通过SDS - PAGE 10确定的P34和P27片段的迁移确认PAK2充分裂解。自身磷酸化条件下的适当核查，是通过在一个单独的使用（32 P）ATP或质谱检测磷酸化的实验的放射自显影。 </li></ol> 2。鉴定胃蛋白酶的消化PAK2片段<ol><li>加入1毫升0.1％Trifluroacetic酸（TFA），在pH 2.5洗两次琼脂糖珠使用前。然后，离心机在2000 XG的样本，以消除珠15秒。 </li><li>添加PAK2（2μL150 mM氯化钠在20微克，50毫米的Tris - HCl和2在pH为7.5毫米的DTT）100μL0.1％TFA，并与胃蛋白酶偶联的琼脂糖珠30μL的样品孵育5分钟。 </li><li>平衡一个反相高效液相色谱法，在pH值2.5 0.1％TFA的溶剂与主系统的C18柱。胃蛋白酶消化（50μL）使用100分钟线性梯度为0 - 80％，含0.2毫升/分钟的流速在0.1％TFA的乙腈洗脱。馏分收集每2分钟。 </li><li>干燥速度VAC（3 H）的高效液相色谱馏分和重悬在乙腈（5μL）和0.1％TFA（5μL）在pH 2.5的解决方案的样品。 </li><li>最初，每个部分使用的MALDI - TOF PerSeptive生物系统公司的航海家德（STR体育碧肽的筛选osystems，福斯特城，加利福尼亚，美国）。 </li><li> 16分数含有肽受到串联质谱使用了Q - TOF质谱仪和一个QSTAR XL oMALDI的MS / MS识别的片段。 </li><li>每个肽的序列是验证与理论的m / z PAK2小学的使用蛋白质勘探者网站（序列为基础的比率由串联质谱<a href="http://prospector.ucsf.edu/">产生</a>的产物离子http://prospector.ucsf.edu/）11。 </li></ol> 3。测量酰胺H / D交换<ol><li>水浴中，保持所有的解决方案和试剂H / D交换需要在25 ° C。 ATP是溶解于水，pH值调整到7.0，在使用前。额外的4mm ATP在D 2 O缓冲区，以防止稀释后的PAK2 ATP的分离。 </li><li>除了​​2μL18μL缓冲D 2 O（50 mM的MOPS pH值6.98，125毫米氯化钠）启动H / D交换（20微克）PAK2包含150毫米的缓冲区，50毫米的Tris - HCl（pH值7.5）和2毫米的DTT，导致在10毫克/毫升的PAK2水平和7.0的pH值在0氯化钠° C。 </li><li>孵育样品一式三份，在H / D交换为0，0.5，1，3，5，10分钟，或24小时</li><li>淬火加入180μL冰冷的0.1％TFA，在pH值2.2，这使在终体积为200μLpH值至2.5的H / D交换过程。对于样品含有ATP，180μL冰冷的0.11％，pH值2.2 TFA是用来保持在淬火条件下的pH值在2.5。 </li><li>等分每个淬火样品（100微升）添加到30μL激活胃蛋白酶偶联的琼脂糖珠。胃蛋白酶的消化允许进行五分钟的冰和样品振荡，每隔30秒。 </li><li>终止于5000 XG样品离心15秒的消化，在4 ° C至删除的胃蛋白酶。 </li><li>迅速冻结于-80 ° C的样品在液态氮和存储不超过2天之前的MALDI - TOF分析。 </li><li>用于MALDI基质为5 mg / ml的α-氰基- 4 -羟基酸（CHCA）溶解在乙腈，乙醇和0.1％TFA在pH 2.5的溶液（1:1:1），在0 ° C举行</li><li>部分迅速解冻每个样品和混合矩阵和4℃的MALDI靶板上的现货与1μL1μL每个样品。 </li><li>应用温和的真空板，干在30秒点之前的MALDI - TOF分析。之间的样品和光谱检索解冻的时间大约是3分钟。 </li><li>收购一个PerSeptive生物系统航海家DE STR MALDI - TOF质谱。在2 GHz的采样率在100,000个数据通道采集数据，与20,000 - V加速电压和65％的电网电压，使用延迟提取180 ns脉冲延迟。一百扫描，平均每2分钟。 MALDI - TOF仪器并不局限于任何品牌或任何特定的软件。 </li><li>使用数据浏览器4.0的计算机程序calcul吃的每一个片段的氘。 </li><li>计算后的交流基础上的实际注册的氘核数量比例在24小时除以所有可能的交换网站，这是除骨干酰胺的N -末端酰胺（ 图 1）H / D交换。 </li></ol> 4。代表性的成果： 通过SDS - PAGE Commassie染色的caspase裂解和自身磷酸化验证。无效PAK2是一个58 kDa的单波段。凋亡的PAK2切割完成，并产生两个片段，P27和P34迁移分开。 autophosphorylated P27和P34的显示，由于完全磷酸化的P27片段（图2）智障迁移的凝胶重叠的迁移。 H / D交换实验进行了多次在0和10分钟（ 图3）之间。作为一个例子，时间当然氘纳入到米/ ZP不活跃的PAK2 1697.85 EAK是随着时间的推移群众信封转变所示的多种同位素峰组成。 <img alt="图1" src="/files/ftp_upload/3602/3602fig1.jpg" /> 图1。公式计算的氘。 <img alt="图2" src="/files/ftp_upload/3602/3602fig2.jpg" /> 图2自身磷酸化和caspase裂解PAK2。 SDS - PAGE和Coommassie蓝染色凋亡裂PAK2（左线），完好无效PAK2（中线）和caspase裂的autophosphorylated PAK2（右线）进行了分析。改编自许，YH等2。 <img alt="图3" src="/files/ftp_upload/3602/3602fig3.jpg" /> 图3。质谱氘注册成立的一个非活动PAK2的时间过程。无效PAK2遭受的H / D exchaNGE为0-10分钟，并用MALDI - TOF分析。在H / D交换的时间当然扩大的MALDI - TOF峰m / z为1697.85同位素分布的一个例子是所示。红色虚线表示平均群众信封和群众信封的转变，显示了一种肽氘。

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. 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没有利益冲突的声明。

胃蛋白酶消化片段的鉴定是关键的一步，H / D交换实验。不正确识别的肽，会导致错误的结论。胃蛋白酶消化PAK2是通过反相高效液相色谱C18柱洗脱获得一个干净的背景。在我们的实验中，共收集了40分数和遭受的MALDI - TOF。多肽，可确定每个分数。肽串联质谱（Q - TOF MS / MS和oMALDI的MS / MS），以确定它们的DNA序列。肽的MS / MS谱图应至少有三个产品离子，以确认鉴定肽。 数据浏览器4.0的计算机程序（Applied Biosystems公司）执行初始基线校正和噪声过滤。必须校准每个频谱基于两个测序峰。我们我们的频谱校正理论质量的undeuterated m / z为923.45和1697.84。 T他质量校准后的精度可以达到10 PPM。单同位素氘后，可能会转移到一个更高的质量。单一步骤增加这些m / z为比值取得与氘增强人民群众。群众信封峰强度不会影响氘水平。然而，在一个特定的质量信封同位素峰的峰强度计算的平均质量的关键。纳入氘核的计算的第一步是从氘样品的非氘样品减去肽质量。其他因素，D 2 O稀释，侧链和背面的交流中残留的氘核，将需要进一步考虑计算（ 图1） 。 D 2 O稀释，混合后的样品和 D 2 O缓冲区启动H / D 交换 ，D 2 O稀释倍数。胃蛋白酶消化的侧链残留氘核（4.5％）版多肽需要减去。背面交换氘氘和胃蛋白酶消化肽质量测量过程中的所有不可避免的损失。 最溶剂的访问后24小时H / D交换肽（M / Z = 1105.60），代表了全氘地区。每个肽纳入氘核的氘和氘非PAK2消化性溃疡肽的质心之间的差异。最高度氘肽的m / z 1105代表全氘PAK2 24小时的H / D交换时。回到外汇比率计算实际纳入氘核H / D交换肽在m / z为1105的所有可能的交换网站分为24小时。

<table border="1"></table><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> D 2 O </td><td>爱秩序</td><td> 7789-20-0 </td><td></td></tr><tr><td> α-氰基-4 - 羟基苯甲酸</td><td>西格玛</td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI - TOF </td><td>体育生物系统公司</td><td>航海家DE海峡</td><td></td></tr><tr><td> Q - TOF质谱仪</td><td> Q - TOF创世纪的全球</td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI的MS / MS </td><td>应用生物系统公司</td><td></td><td></td></tr><tr><td>数据浏览器4.0的计算机程序</td><td>应用生物系统公司</td><td></td><td></td></tR&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

酰胺氢/氘交换质谱（H / D交换）已被广泛用于分析蛋白质 - 蛋白质相互作用，蛋白质构象变化，蛋白质动力学，蛋白质 - 配体相互作用的接口。 H / D交换骨干酰胺的位置已经被用来测量质谱 1,2,3中的一种蛋白质的微区的氘率。这种方法的分辨率取决于利益氘蛋白的胃蛋白酶消化成肽，通常范围从3-20残留。虽然H / D交换质谱测量的分辨率比单残留的异核单量子相干（HSQC）核磁共振，在H / D交换的质谱测量方法测量的分辨率较低的大小并不局限于蛋白4。 H / D交换是在水溶液中，维持蛋白质构象进行了。我们提供了一种方法，UTILizes 2检测的MALDI - TOF，而不是一个HPLC / ESI（电喷雾电离） - MS系统5,6。 MALDI - TOF提供消化的蛋白质肽的精确质量强度数据，在这种情况下蛋白激酶PAK2（也称为γ-白）。蛋白水解白2进行脱机胃蛋白酶的消化。这另一种方法，当用户不具有访问一个连接到质谱高效液相色谱法和胃蛋白酶列，或当HPLC法测定胃蛋白酶列不会导致在一个最佳的消化地图，例如，大量二硫化物保税分泌型磷脂酶A 2 （SPLA 2）。利用这种方法，我们成功地监测在PAK2激活caspase 3的裂解和自身磷酸化7,8,9氘水平的变化。

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

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

成功地利用MALDI - TOF质谱监察PAK2活化蛋白激酶的酰胺氢/氘交换。

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 ...

amide-hydrogendeuterium-exchange-maldi-tof-mass-spectrometry-analysis

Research

JoVE Journal

Biology

17.7K 次观看.  Tunghai University. 成功地利用MALDI - TOF质谱监察PAK2活化蛋白激酶的酰胺氢/氘交换。

视频: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

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 ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

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 ...

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 ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...