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. 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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 & 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

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

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

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

科研

化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

生物化学

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

原位近接结扎法检测蛋白异构体的 Heterodimerization

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

癌症研究

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

免疫沉淀法研究受体与酶的功能相互作用

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

免疫与感染

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

摘要

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此视频中的章节

使用氢交换质谱分析蛋白质动力学

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

用放射性标记的ATP测定蛋白激酶活性

原位近接结扎法检测蛋白异构体的 Heterodimerization

通过 SHP2 （PTPN11）磷酸酶抑制剂评估细胞目标参与度

使用新激酶蛋白的强生化方法在分子水平上进行表征

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

免疫沉淀法研究受体与酶的功能相互作用

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

摘要

探索更多视频

此视频中的章节

使用氢交换质谱分析蛋白质动力学

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

用放射性标记的ATP测定蛋白激酶活性

原位近接结扎法检测蛋白异构体的 Heterodimerization

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

使用新激酶蛋白的强生化方法在分子水平上进行表征

氢-铀交换质谱仪（HDX-MS）平台，用于研究肽生物合成酶

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

免疫沉淀法研究受体与酶的功能相互作用

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

通过 SHP2 （PTPN11）磷酸酶抑制剂评估细胞目标参与度

可视化蛋白质激酶在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性