This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).

注:此协议为减少与干滴法制备麦芽三糖和激肽片段（1-7）的空间异质性的发展。该协议包括三个主要步骤，包括准备和预处理，样品沉积和干燥，和质谱数据分析。的程序概述，并在下面更详细描述: 1.准备和预<ol><li>清洗样品板<ol><li>戴上丁腈手套，并用清洁剂和蒸馏水，去离子水（DDW）轻轻手洗样品板。 </li><li>冲洗用甲醇（MeOH）和DDW样品板。 </li><li>插入样品板在一个600ml烧杯中，用DDW填充。 </li><li>超声处理在DDW样品板为在超声​​浴中（200瓦，40千赫）15分钟。 </li><li>从烧杯中取出DDW和填充用甲醇烧杯中。 </li><li>超声处理在MeOH样品板在超声浴（200瓦，40千赫）15分钟。 </li><li>吹掉溶剂滴上用氮气板和保持样品板样品沉积之前干燥。 </li></ol></li><li>调节烘干室温度 注意:干燥室是一个35×20×45 立方厘米 （宽x深x高）丙烯酸室图1显示了干燥系统的图片。该腔室通过气体流量计以恒定的流率通入室温的氮气，以保持由所述干燥室内部安装了一个校准湿度计监测的低相对湿度条件。在装有编程的恒温循环器的干燥室的铜基块用于容纳不锈钢样品板。铜基块能够从5样品板温度调节至25℃。空气，铜基块和样品板的温度由K型热电偶监控。 <ol><li>打开门，迅速投入样品板的铜基块然后关门。 </li><li>手动调节气体流量计来设置氮气流速至10标准立方英尺每小时（SCFH）。 </li><li>监测由湿度计和微调气体流量计在干燥室中的相对湿度，以确保相对湿度总是低于25％。 </li><li>监测样品板的由K型热电偶的温度和手动调整水循环器温度，直到该样品板达到5℃下的实验或用于控制室温（25℃）。 注意:为了在一个设计温度稳定的样品板，水环温度通常设定为0〜小于设计的样品低5℃。例如，为了保持5℃，在样品板中，水环行器的温度设定在0〜2℃的范围内;保持样品板在25℃，水环行器的温度设定在23至25℃的范围内。 </li><li>确保所需的温度和相对湿度样品沉积之前达到（ 表1）。 注:所有参数，以及用于与不同的样品板的温度的干燥方法其设置值示于表1。 注:在一个较低的样品板温度，如果室内大门是敞开的很长一段时间，可能会发生在样品板上冷凝水。如果出现冷凝水，关上门，直到水冷凝干燥就可以了不要存放任何样本。 </li></ol></li><li>矩阵和分析物溶液的制备<ol><li>基质溶液的制备<ol><li>制备0.1，用50％乙腈（ACN）M THAP溶液:50％DDW水溶液。 </li></ol></li><li>分析物的制备<ol><li>准备10 -4与DDW中号麦芽三糖的解决方案。 </li><li>在50％乙腈制备10 -5 M激肽片段（1-7）溶液（ACN）:50％DDW水溶液。 </li></ol></li></ol></li></ol> 2.样品沉积和干燥<ol><li>预混物的0.1M THAP溶液0.25微升和0.25微升10 -4 M麦芽三糖或10 -5 M激肽片段（1-7）在微量离心管的解决方案。 </li><li>涡3秒的混合溶液中。 </li><li>离心2秒（2,000 XG）的混合溶液中，收集在离心管底部的溶液。 </li><li>打开干燥室的门，小心地存放0.1微升与吸管样品板解决方案，并立即关门。 </li><li>等待样品液滴干燥。 注:本典型地观察到的干燥时间具有不同的样品板的温度列于表1对于5℃的样品板的温度，在平均干燥时间为800〜1000秒;为25℃的样品板的温度，在平均干燥时间为100〜150秒EC。 </li><li>干燥后，打开干燥室的门。 </li><li>设置水循环器温度至室温（25℃）。 注意:如果该样品板是不断在室温（25℃）在干燥过程中保持跳过此步骤。 </li><li>该样品板温度恢复到室温（25℃）后，从干燥室的样品板。 </li><li>检查5倍的立体显微镜下样品形态和拍摄快照亮场图像。 注意:如果晶体形态并不如预期，有必要用相同的程序准备一个新的样品。典型的晶体形貌示于图2的上面板。 注意:在低的样品板的温度，如5℃的情况下，它预热样品板来带离干燥室的前室的温度很重要。当沉积的样品中， 不守预混搜索解决方案N的吸管在10秒内一角。 不要沉积样品后，再次使用预混液。 图2的上图显示具有不同的样品板的温度制备的样品的亮场图像。 </li></ol> 3.质谱数据分析<ol><li>质谱数据采集 注:制备后，将样品可以利用成像质谱进行分析。在目前的研究中，成像MS实验使用的是实验室自制同步的双极性TOF进行（DP-TOF）成像质谱仪。15商业MALDI-TOF质谱仪具有成像能力也适用于这样的实验。质谱仪以线性萃取并用优化的提取延迟正离子模式操作。离子的动能为20千伏。激光束尺寸为35微米的样品表面上的直径，并且每一个点的光谱是大街5激光发射愤怒。 <ol><li>插入样品板进入MALDI质谱仪。 </li><li>执行成像质谱分析，以在步骤2.1-2.9中制备的样品。 </li><li>选择在结果窗口所示的质量列表的特征质量峰并点击"2D"绘制二维离子的图像。 注:对于麦芽三糖与THAP混合的特征峰sodiated麦芽三糖，质子THAP和sodiated THAP。用于与THAP混合激肽片段（1-7），特征峰包括质子激肽片段（1-7），质子化THAP和sodiated THAP。 </li><li>点击弹出窗口中的调节按钮，以确定信号强度的上限和下限，并点击"保存图片"。此设置定义离子图像的对比度。 注意:在每个单独的数据集，裂化区域和示出低亮度的零点被消除。 </li><li>观察比较离子与采取了一步2.9亮场图像的图像。 注:成像质谱和特定离子的图像的构造可以与商业仪器来实现。由于各种数据采集和分析软件时，用户应遵循由仪器供应商提供，以获得高品质的图像的软件指令。 </li></ol></li><li>数据分析 注:样品的异质性定量分析。在这个演示中，每个样本分为内部开发的分析离子的空间分布由软件的多个同心区。也可以使用单独的数据分析软件进行分析。 <ol><li>点击在结果窗口中显示的离子图像中的空斑和裂化区以除去不重要的区域。 注:此过程定义离子形象的重要领域。 </li><li>单击"查找边缘"按钮找到离子图像的最外层。 </li><li>点击"扣除"保存在数据库中最外层的离子丰度的信息和同时除去从离子图像这一层。这代表最外层一个复选框会出现在结果窗口中的"输出数据"名单。 </li><li>重复步骤3.2.2和3.2.3直到离子图像的中心被定义。 </li><li>点击并在"输出数据"列表中选择所有的复选框，然后单击"导出"导出数据。 </li><li>打开使用电子表格软件来计算每一层的平均离子丰度来获得离子的空间分布信息导出的数据。 </li></ol></li></ol>

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The authors declare no competing financial interest.

根据以往的理论预测，液滴内温度引起的水动力流可以向外克服溶剂挥发诱导毛细血管流动。当温度液滴增加内梯度分子的这种内部再循环的效率提高。根据预测结果，保持样品板温度时低于5℃，同时保持其周围环境温度，再循环流动的液滴内的平均流速大于向外毛细管流动的速度的4倍左右。如果样品板温度是相同的环境中，再循环流的平均速度比向外毛细流慢1800倍。这一计算的结果表明，样?...

Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the "sweet spot" problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.

<table border="0" cellpadding="0" cellspacing="0" width="787">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Reagent</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent powder</td>
			<td style="width:157px;">Alconox</td>
			<td style="width:139px;">242985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">106009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Acetonitrile</td>
			<td style="width:157px;">Merck</td>
			<td style="width:139px;">100003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">2,4,6-trihydroxyacetophenone (THAP)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">T64602&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bradykinin fragment (1-7)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">B1651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Maltotriose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:139px;">47884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette tips</td>
			<td style="width:157px;">Mettler Toledo</td>
			<td style="width:139px;">17005091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microcentrifuge tube</td>
			<td style="width:157px;">Axygen</td>
			<td style="width:139px;">MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Milli-Q water purification system</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:139px;">ZMQS6VFT1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powder-free nitrile gloves</td>
			<td style="width:157px;">Microflex</td>
			<td style="width:139px;">SU-690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">600 mL beaker</td>
			<td style="width:157px;">Duran</td>
			<td style="width:139px;">2110648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasonic cleaner</td>
			<td style="width:157px;">Delta</td>
			<td style="width:139px;">DC300H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hygrometer</td>
			<td style="width:157px;">Wisewind</td>
			<td style="width:139px;">5330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nitrogen gas flowmeter</td>
			<td style="width:157px;">Dwyer</td>
			<td style="width:139px;">RMA-6-SSV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">K-type thermocouples</td>
			<td style="width:157px;">Digitron</td>
			<td style="width:139px;">311-1670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Centrifuge</td>
			<td style="width:157px;">Select BioProducts</td>
			<td style="width:139px;">Force Mini&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette</td>
			<td style="width:157px;">Rainin</td>
			<td style="width:139px;">pipet-lite XLS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereomicroscope</td>
			<td style="width:157px;">Olympus</td>
			<td style="width:139px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">MALDI-TOF stainless steel sample target</td>
			<td style="width:157px;">this lab</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of homogeneous maldi samples for quantitative applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

亮场图像以及以5样品板温度和25℃制备麦芽三糖和激肽片段（1-7）的所述MS图像示于图1中 。在sodiated麦芽三糖，的离子信号主要填充的情况下在样品区域的周围时，它是在25℃的样品板温度制备。通过降低样品板温度至5℃，信号均匀在整个样品区填充。在5℃，准备样品时，唯一明显的缺点是，有比在25℃制备的样品更多的裂缝。质子缓激肽片段（1-7）的离子?...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

一种通过调节过程中样品干燥过程中衬底温度降低MALDI质谱离子信号的空间分异协议证明。

定量应用同质MALDI样品的制备

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Research

JoVE Journal

Biochemistry

8.7K 次观看.  Academia Sinica. 一种通过调节过程中样品干燥过程中衬底温度降低MALDI质谱离子信号的空间分异协议证明。 

视频: Preparation of Homogeneous MALDI Samples for Quantitative Applications

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

高密度脂蛋白的分离的非编码小RNA定量

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

科研

生物化学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

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, ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

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 ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

通过并行反应监测进行无处不在的链分析

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

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 ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

EWS-FLI1凝聚物在DNA上组装的单分子成像

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...