作者感谢罗伯特·舒斯特（Robert Schuster）在数据验证方面的帮助。

mAbScale 的高级工作流程 如图 2 所示。每个步骤都有更复杂的内部决策分支、循环和组合函数。描述计算过程的详细算法工作流程见 补充图 1。应用程序输出以电子表格格式保存在用户选择的文件夹中。输出文件由多个单独的工作表组成，这些工作表可以归类为用户输入、分子量计算和平均同位素质量数推导的参考（补充表中提供了示例输出）。用户输入的工作表包括用户输入的蛋白质氨基酸序列和其他信息、平均元素质量和聚糖质量，用于计算元素组成和不同分子量。分子量计算表包括各种形式的化学组成、有和没有糖基化和化学修饰的还原质量数，以及有和没有糖基化和化学修饰的完整质量数。如果用户在用户输入页面中输入两种不同的 HC 和/或两种不同的 LC，则将自动生成含有半抗体质量的纸，因为半抗体是需要相对于所需异二聚体进行鉴定和定量的主要杂质。mAbScale 的源代码可通过以下存储库访问:https://github.com/kkhatri99/mAbScale。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65298/65298fig02.jpg"> 图 2:使用该应用计算元素组成和质量所涉及的步骤概述。 颜色编码可用于链接到 补充图1中描述的工艺流程。 <a href="https://www.jove.com/files/ftp_upload/65298/65298fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
1. 打开 mAbscale 应用程序
<ol><li>双击可执行文件的图标打开软件应用程序。</li>
</ol>2. 序列输入
<ol><li>在标有 1 的相应文本框中输入重链和轻链序列，不带任何空格。<ol><li>对于 bsAbs，在标记为 2 的第二组文本框中添加其他重链或轻链。对于具有相同重链和轻链的mAb，留 2 个空白。</li>
		<li>选中 N-Terminal Cyclization 和/或 C-Terminal Clipping 复选框（如果这些重链端子变体适用）。</li>
		<li>将任何化学修饰（包括ADC分子的接头和有效载荷）添加到"重链化学模组"和/或"轻链化学模组"文本框中。<ol><li>将修饰指定为元素组成，例如 CaCl2。修饰将被添加到相应的蛋白质亚基或链中。 注:化学成分也可以通过在元素组成前面加上-符号来从亚基或链中减去。例如，-H2O 将从亚基组成和质量中减去水分子。</li>
		</ol></li>
	</ol></li>
</ol>3. 指定二硫键的数量
<ol><li>在标记为"二硫 化物总数"的文本框中指定蛋白质分子中二硫键的数量。</li>
	<li>在未还原的 HC 二硫 化物文本框中输入未还原的 HC 二硫化物的数量，在未还原的 LC 二硫化物文本框中输入未还原的 LC 二硫 化物的数量，具体取决于还原程度（完全还原与部分还原）。 注:mAb亚基的简化质量数分析涉及二硫键连接的重链和轻链的还原/分离。</li>
	<li>如果 mAb 轻链上存在糖基化，请选中 Light Chain is Glycosylated 复选框。</li>
</ol>4. 设置输出文件夹并运行应用程序
<ol><li>单击" 浏览 "按钮为"输出文件夹"文本框选择 输出文件夹 。</li>
	<li>在 Excel 文件（无分机） 文本框中输入不带文件扩展名的输出文件名（自动另存为 .xlsx）。</li>
	<li>单击 "提交 "按钮开始应用程序。输出文件可以在指定的文件夹中找到。 注:元素质量和聚糖列表可以通过分别编辑带分隔符的文本文件 Element_Mass.csv （补充编码文件 1）和 聚糖.csv （补充编码文件 2）来定制。这些文件必须与 mAbScale.exe （补充编码文件 3）可执行文件放在同一文件夹中，应用程序才能执行。应用程序将在执行一次后自动关闭。如果需要第二次计算，用户将不得不再次启动应用。</li>
</ol>

基于 Python 的生物治疗药物实体精确质量计算工具

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该软件在 Apache 2.0 许可下发布。葛兰素史克研究与发展有限公司版权所有 （2022）。保留所有权利。根据 Apache 许可证 2.0 版（以下简称"许可证"）获得许可;除非遵守许可，否则您不得使用此文件。您可以在 http://www.apache.org/licenses/LICENSE-2.0 获取许可证的副本。除非适用法律要求或书面同意，否则根据许可分发的软件按"原样"分发，不附带任何明示或暗示的保证或条件。请参阅许可证，了解许可证下管理权限和限制的特定语言。L.C.是葛兰素史克（GSK）的员工。T.H. 和 K.K. 作为 GSK 的员工开发了该软件，现在分别是 Merck 和 Moderna 的合作伙伴。

mAbScale提供了一个直观的用户界面，可以灵活地改变质量数和元素计算的构建模块。用户应对目标分子有基本的了解，以便使用该应用程序，得出正确的质量数，并解释结果。例如，由于完整或减少质量数的行数众多，完整或减少的质量输出表可能会让人不知所措，因为默认的聚糖数据库包含88种N-连接聚糖，这些聚糖通常存在于治疗性抗体的Fc部分，并且该应用程序计算了数据库18</s...

在过去的二十年里，生物治疗药物已经发展成为现代制药工业的中流砥柱。SARS-CoV2 大流行和其他危及生命的疾病进一步增加了对更快、更广泛地开发生物制药分子的需求 1,2,3。
生物治疗药物分子量与其他分析检测相结合，对于分子的鉴定至关重要。完整和减少的亚基质量数用于整个发现和开发生命周期，作为旨在保持质量的控制策略的一部分，如QTPP（质量目标产品简介）4中所述。
生物制药行业的分析开发在很大程度上依赖于质量测量，以使用肽图分析或多属性方法（MAM）监测进行完整质量分析和深度表征。利用现代质谱 （MS） 平台的这些技术的核心是能够提供高分辨率的精确质量 （HR/AM） 测量。大多数HR/AM仪器的质量精度在0.5-5 ppm范围内，与质量范围成正比。准确测量完整大分子质量数的能力能够快速、可靠地鉴定大分子治疗药物。由于使用大分子（>10 kDa）的典型实验条件无法获得同位素分辨率，因此必须计算平均质量数以进行比较和鉴定5,6。
典型的完整或亚基蛋白质质谱图代表了整体蛋白型图谱，其中包含翻译后修饰 （PTM） 产生的各种分子形式的复合信息以及任何一级结构差异，例如片段或序列变体。这些测量相对容易和高通量的特性使它们对表征和作为过程监测控制具有吸引力7,8。这些实验的数据分析通常要求用户定义分子形式（PTM或其他分子形式的范围）的搜索空间。对于糖基化蛋白，这种搜索空间很大程度上是由糖型异质性驱动的。多个 PTM、二硫键构型以及沿一级结构的其他变化的组合使得计算所有可能的分子形式成为一项繁琐的任务。因此，手动计算可能的分子形式是一个耗时、耗费资源的过程，极有可能出现人为错误。
在这里，我们提出了一种质量计算工具，该工具是考虑到生物治疗分子的最重要特征而开发的，例如mAb、bsAb、ADC等。该工具允许轻松合并搜索空间变量，以实现质量和元素组成的一致计算。该工具的模块化特性将使其能够进一步发展并应用于其他模式的质量计算和质量匹配。
GUI模块允许用户指定质量计算的输入，如图 1所示;具体来说，用户输入轻抗体链和重抗体链的单字母氨基酸序列。重链 N 端环化和 C 端赖氨酸剪切的常见修饰作为复选框包括在内。此外，可以通过相应的 Chem Mod 文本框从这些蛋白质链中添加/减去化学式/元素组成。这允许用户灵活地添加元素组成，包括多个翻译后修饰或ADC中的小分子有效载荷。由于大多数治疗性单克隆抗体被设计为去除轻链中的糖基化位点，因此轻链中的糖基化是可选的，可以使用 GUI 上的复选框进行指定。
抗体完整质量数分析的典型变化是亚基质量数分析，其中轻链通过减少链间二硫键从重链中分离出来。根据所用还原剂的强度，链内二硫键可能被裂解，也可能不被切割。用户可以根据IgG亚型或半胱氨酸偶联ADC9灵活地输入二硫键的总数。
该应用程序以自下而上的方式计算质量，其中首先计算单个重链和轻链的元素组成。接下来，通过调整计算出的元素组成来解释重链（HC）N端环化Lys-clipping。然后将任何指定的化学修饰应用于重链和/或轻链。根据分析类型和用户指定的二硫键模式，调整两条多肽链的氢数量。糖基化的HC和轻链（LC）（可选）质量数是根据用户的输入计算的。最后，将多个HC和LC质量数合并，并自动更新二硫键数以计算完整质量数。
对于较大的分子（如完整的蛋白质），当使用具有典型分辨能力的质谱仪时，由于加性质量缺陷，无法测量单同位素质量数。取而代之的是，测量或报告标称或平均质量 5,10,11,12,13。平均元素质量可以根据用于策划质量的来源而变化14,15。虽然元素质量的差异可能很小，但对于大分子分子量计算来说，它们加起来可以产生显著的值。软件应用程序中默认使用的平均元素质量如附表1所示。对于生物制药研发（R&D）领域等受监管的环境，保持一致的分子量非常重要，因为质量的变化可能意味着在监管备案期间分子实体发生变化。为了实现元素质量使用的一致性，软件工具中包含了元素质量字典，作为逗号分隔值 （csv） 文本文件:Element_Mass.csv（补充编码文件 1）。同样，包括mAb上常见的聚糖组合物的精选列表:聚糖.csv（补充编码文件2）。这两个文件都保存在与可执行应用程序相同的文件夹位置，用户可以修改以使用特定的元素质量数列表或聚糖库。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65298/65298fig01.jpg"> 图 1:mAbScale 应用程序的 GUI 界面。 GUI模块允许用户指定质量计算的输入。用户输入轻抗体链和重抗体链的单字母氨基酸序列。重链 N 端环化和 C 端赖氨酸剪切的常见修饰作为复选框包含在内。化学式/元素组成可以通过相应的 Chem Mod 文本框进行添加/减去。 <a href="https://www.jove.com/files/ftp_upload/65298/65298fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acquity UPLC system&#160;</td>
			<td>Waters Corp., Milford, MA</td>
			<td>N/A</td>
			<td>Modular system</td>
		</tr>
		<tr>
			<td>Antibody-drug conjugate (ADC)</td>
			<td>GlaxoSmithKline</td>
			<td>N/A</td>
			<td>Proprietory molecule</td>
		</tr>
		<tr>
			<td>BEH 200 SEC column&#160;</td>
			<td>Waters Corp., Milford, MA</td>
			<td>176003904</td>
			<td></td>
		</tr>
		<tr>
			<td>Bispecific mAb</td>
			<td>GlaxoSmithKline</td>
			<td>N/A</td>
			<td>Proprietory molecule</td>
		</tr>
		<tr>
			<td>Byos</td>
			<td>Protein Metrics, Cupertino, CA</td>
			<td>https://proteinmetrics.com/byos/ 
			Version 4.5</td>
			<td></td>
		</tr>
		<tr>
			<td>GPMAW</td>
			<td>GPMAW</td>
			<td>http://www.gpmaw.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS grade water&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>W6-1</td>
			<td></td>
		</tr>
		<tr>
			<td>mAb standard&#160;</td>
			<td>Waters Corp., Milford, MA</td>
			<td>186009125</td>
			<td>Waters Humanized mAb Mass Check Standard</td>
		</tr>
		<tr>
			<td>mAbScale</td>
			<td>GlaxoSmithKline</td>
			<td>Apache License, Version 2.0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Xevo G2 Q-TOF mass spectrometer</td>
			<td>Waters Corp., Milford, MA</td>
			<td>N/A</td>
			<td>Modular system</td>
		</tr>
	</tbody>
</table>

an open-source framework for mass calculation of antibody-based therapeutic molecules

生物治疗肿块是验证身份和结构完整性的一种手段。完整蛋白质或蛋白质亚基的质谱 （MS） 为生物制药开发的不同阶段提供了一种简单的分析工具。当MS的实验质量数在理论质量数的预定义质量数误差范围内时，蛋白质的身份得到确认。虽然有几种计算工具可用于计算蛋白质和肽分子量，但它们要么不是为直接应用于生物治疗实体而设计的，要么由于付费许可证而存在访问限制，要么需要将蛋白质序列上传到主机服务器。 
我们开发了一种模块化质量数计算程序，可以轻松测定治疗性糖蛋白（包括单克隆抗体 （mAb）、双特异性抗体 （bsAb） 和抗体-药物偶联物 （ADC））的平均或单同位素质量数和元素组成。这个基于 Python 的计算框架的模块化特性将允许该平台在未来扩展到其他模式，例如疫苗、融合蛋白和寡核苷酸，并且该框架也可用于自上而下的质谱数据查询。通过创建具有图形用户界面 （GUI） 的开源独立桌面应用程序，我们希望克服在无法将专有信息上传到基于 Web 的工具的环境中使用的限制。本文介绍了mAbScale工具在不同基于抗体的治疗方式中的算法和应用。

选择多种单克隆抗体来代表不同类型的单克隆抗体。选择市售的mAb标准品来表示在Fc区域具有相同重链、相同轻链和一个N-连接糖基化位点的常规mAb。此外，还选择了具有额外轻链 N-连接糖基化的单克隆抗体、双特异性单克隆抗体和抗体-药物偶联物 （ADC） 单克隆抗体，以扩大应用范围。 表1总结了这些示例mAb的化学成分、计算质量数、测量质量和质量数误差。mAbScale报告的蛋白质化学...

Watch this Scientific Journal Video about Advancing Biotherapeutic Mass Calculation by Introducing mAbScale, a Python-Based Desktop Application at JoVE.com

作者聚焦:通过推出基于 Python 的桌面应用程序 mAbScale 推进生物治疗药物质量计算

本文介绍了如何使用软件应用程序mAbScale来计算基于单克隆抗体的蛋白质疗法的质量数。

用于基于抗体的治疗分子质量数计算的开源框架

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides ...

对大蛋白质分子进行精确质量数计算的挑战是与测量相关的质量缺陷。因此，只能测量平均质量。微小的元素质量差异可能会累积，导致大的辅助治疗分子（如单克隆抗体）出现显着差异。我们的工具明确解决了基于抗体的疗法（如 MAP、双特异性和抗体药物偶联物）在一致质量数计算方面的挑战。由于涉及专有信息，商业生物制药开发领域的研究人员不得不依赖手动计算或不依赖于与外部服务器通信的工具。现有工具要么需要许可证，要么没有考虑到生物制药开发的特定需求。MapScale为用户和开发人员提供了用户友好的界面和适应性强的开源框架。它支持维护多个版本的元素质量和修饰列表，并允许列表共享以保持计算的一致性。可以使用内部服务器或基于云的部署在企业级别共享代码，并具有受控访问以保护专有信息。

an-open-source-framework-for-mass-calculation-antibody-based

Research

JoVE Journal

Biochemistry

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules

1.4K 次观看.  GlaxoSmithKline. 对大蛋白质分子进行精确质量数计算的挑战是与测量相关的质量缺陷。因此，只能测量平均质量。微小的元素质量差异可能会累积，导致大的辅助治疗分子（如单克隆抗体）出现显着差异。我们的工具明确解决了基于抗体的疗法（如 MAP、双特异性和抗体药物偶联物）在一致质量数计算方面的挑战。由于涉及专有信息，商业生物制药开发领域的研究人员不得不依赖手动计...

视频: 作者聚焦:通过推出基于 Python 的桌面应用程序 mAbScale 推进生物治疗药物质量计算

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. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Microcrystal Electron Diffraction of Small Molecules

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been developed to solve structures of proteins and small molecules using standard electron cryo-microscopy (cryo-EM) equipment. In this way, small molecules, peptides, soluble proteins, and membrane proteins have recently been determined to high resolutions. Protocols are presented here for preparing grids of small-molecule pharmaceuticals using the drug carbamazepine as an example. Protocols for screening and collecting data are presented. Additional steps in the overall process, such as data integration, structure determination, and refinement are presented elsewhere. The time required to prepare the small-molecule grids is estimated to be less than 30 min.

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

小分子的微晶电子衍射

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...