Name: preparation of homogeneous maldi samples for quantitative applications
Uploaded: 2016-10-28T13:00:00.000+00:00
Duration: 8 min 1 s
Description: Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

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>

<ol>
	<li>Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Desorption+Ionization+of+Proteins+with+Molecular+Masses+Exceeding+10000+Daltons.">Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons.</a> Anal. Chem. 60, 2299-2301 (1988).</li><li>Beavis, R. C., 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=Velocity+Distributions+of+Intact+High+Mass+Polypeptide+Molecule+Ions+Produced+by+Matrix+Assisted+Laser+Desorption.">Velocity Distributions of Intact High Mass Polypeptide Molecule Ions Produced by Matrix Assisted Laser Desorption.</a> Chem. Phys. Lett. 181, 479-484 (1991).</li><li>Beavis, R. C., Chaudhary, T., 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=Alpha-Cyano-4-Hydroxycinnamic+Acid+as+a+Matrix+for+Matrix-Assisted+Laser+Desorption+Mass-Spectrometry.">Alpha-Cyano-4-Hydroxycinnamic Acid as a Matrix for Matrix-Assisted Laser Desorption Mass-Spectrometry.</a> Org. Mass Spectrom. 27, 156-158 (1992).</li><li>Ehring, H., Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Photoionization+and+Photochemistry+in+Ionization+Processes+of+Organic-Molecules+and+Relevance+for+Matrix-Assisted+Laser+Desorption+Ionization+Mass-Spectrometry.">Role of Photoionization and Photochemistry in Ionization Processes of Organic-Molecules and Relevance for Matrix-Assisted Laser Desorption Ionization Mass-Spectrometry.</a> Org. Mass Spectrom. 27, 472-480 (1992).</li><li>Strupat, K., Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,5-Dihydroxybenzoic+Acid+-+a+New+Matrix+for+Laser+Desorption+Ionization+Mass-Spectrometry.">2,5-Dihydroxybenzoic Acid - a New Matrix for Laser Desorption Ionization Mass-Spectrometry.</a> Int. J. Mass Spectrom. Ion Process. 111, 89-102 (1991).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaporation+of+a+Sessile+Droplet+on+a+Substrate.">Evaporation of a Sessile Droplet on a Substrate.</a> J. Phys. Chem. B. 106, 1334-1344 (2002).</li><li>Deegan, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Flow+as+the+Cause+of+Ring+Stains+from+Dried+Liquid+Drops.">Capillary Flow as the Cause of Ring Stains from Dried Liquid Drops.</a> Nature. 389, 827-829 (1997).</li><li>Hu, J. -. B., Chen, Y. -. C., Urban, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coffee-Ring+Effects+in+Laser+Desorption/Ionization+Mass+Spectrometry.">Coffee-Ring Effects in Laser Desorption/Ionization Mass Spectrometry.</a> Anal. Chim. Acta. 766, 77-82 (2013).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Tissue+Analysis+Using+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry:+Practical+Aspects+of+Sample+Preparation.">Direct Tissue Analysis Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Practical Aspects of Sample Preparation.</a> J. Mass Spectrom. 38, 699-708 (2003).</li><li>Hu, H., Larson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marangoni+Effect+Reverses+Coffee-Ring+Depositions.">Marangoni Effect Reverses Coffee-Ring Depositions.</a> J. Phys. Chem. B. 110, 7090-7094 (2006).</li><li>Bhardwaj, R., Fang, X., Attinger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+Formation+During+the+Evaporation+of+a+Colloidal+Nanoliter+Drop:+A+Numerical+and+Experimental+Study.">Pattern Formation During the Evaporation of a Colloidal Nanoliter Drop: A Numerical and Experimental Study.</a> New J. Phys. 11, 075020 (2009).</li><li>Savino, R., Paterna, D., Favaloro, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buoyancy+and+Marangoni+Effects+in+an+Evaporating+Drop.">Buoyancy and Marangoni Effects in an Evaporating Drop.</a> J Thermophys Heat Tr. 16, 562-574 (2002).</li><li>Probstein, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Tension. in Physicochemical Hydrodynamics : An Introduction. , 305-361 (1994).</li><li>Lai, Y. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Spatial+Heterogeneity+of+MALDI+Samples+with+Marangoni+Flows+During+Sample+Preparation.">Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.</a> J. Am. Soc. Mass Spectrom. 27, 1314-1321 (2016).</li><li>Hsiao, C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Molecular+Imaging+of+Photolabile+Surface+Samples+with+Synchronized+Dual-Polarity+Time-of-Flight+Mass+Spectrometry.">Comprehensive Molecular Imaging of Photolabile Surface Samples with Synchronized Dual-Polarity Time-of-Flight Mass Spectrometry.</a> Rapid Commun. Mass Spectrom. 25, 834-842 (2011).</li><li>Vorm, O., Roepstorff, P., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Resolution+and+Very+High-Sensitivity+in+MALDI+TOF+of+Matrix+Surfaces+Made+by+Fast+Evaporation.">Improved Resolution and Very High-Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation.</a> Anal. Chem. 66, 3281-3287 (1994).</li><li>Gabriel, S. J., Schwarzinger, C., Schwarzinger, B., Panne, U., Weidner, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Segregation+as+the+Major+Cause+for+Sample+Inhomogeneity+in+MALDI+Dried+Droplet+Spots.">Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots.</a> J. Am. Soc. Mass Spectrom. 25, 1356-1363 (2014).</li><li>Mampallil, D., Eral, H. B., van den Ende, D., Mugele, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Evaporating+Complex+Fluids+through+Electrowetting.">Control of Evaporating Complex Fluids through Electrowetting.</a> Soft Matter. 8, 10614-10617 (2012).</li></ol>

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，样品制备过程中减少样品板温度是有利的。实验观察同意这一预测。 样品板脾气ATURE应在整个样品制备过程。 表1中可以精确控制显示了典型的液滴的干燥时间，在不同的样品板上的温度0.1微升的样品。在板沉积样品溶液之前，重要的是要确保在样品板表面干燥。如果准备在低温下的样品时，会出现冷凝水，不建议样品溶液沉积，因为水珠凝结扩大样本地区和稀释的解决方案。因此，为了保持在干燥室的相对湿度在25％以下是重要的。此外，在低温下制备的样品时，该样品板应带离干燥室之前被预热到室温。虽然样品结晶完成后轻微水缩合不改变样本总体，显著缩合应该避免。 使用新鲜预混解决方案是RECOM谁料。一旦预混合溶液暴露于空气中，发生样品溶液的预结晶和最终晶体尺寸和形态可以改变。因此，在移液过程应以合理的效率进行，通常在10秒内，以防止移液管尖内从预结晶的样品液滴。建议观察显微镜下样品的形态，以确保合适的晶体形态是质谱分析之前生产的。如果晶体形态是不如预期的那样，重复必要在沉积过程。 根据我们的理论和实验研究，制备样品在环境条件下安装在低温样品板大大提高在MALDI-MS中的数据再现性和质量。随后的实验也显示信号强度的显著增强这种样品制备方法。用t获得的实验数据他的方法显着提高的MALDI质谱进行定量分析的可靠性。在涉及溶液成分或表面属性更改其它方法相比，8,16-18改变干燥条件是常规的样品更简单，更普遍适用。因此，大多数质谱用户可以在常规的应用从中受益。 改善MALDI信号均匀性与降低样品板温度也是有效的一些其他流行矩阵。例如，改进的α环糊精（α-CD）配THAP，α氰基-4-羟基肉桂酸（CHCA），为低温样品干燥条件下的矩阵信号同质最近已经报道14与改变的样品板的缺点温度是，该方法是目前不适合高通量分析，由于在低温条件下，长的样品的干燥时间。

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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Detergent powder</td><td>Alconox</td><td>242985</td><td></td></tr><tr><td>Methanol</td><td>Merck</td><td>106009</td><td></td></tr><tr><td>Acetonitrile</td><td>Merck</td><td>100003</td><td></td></tr><tr><td>2,4,6-trihydroxyacetophenone (THAP)</td><td>Sigma-Aldrich</td><td>T64602&amp;nbsp;</td><td></td></tr><tr><td>Bradykinin fragment (1-7)</td><td>Sigma-Aldrich</td><td>B1651</td><td></td></tr><tr><td>Maltotriose</td><td>Sigma-Aldrich</td><td>47884</td><td></td></tr><tr><td>Pipette tips</td><td>Mettler Toledo</td><td>17005091</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Milli-Q water purification system</td><td>Millipore</td><td>ZMQS6VFT1</td><td></td></tr><tr><td>Powder-free nitrile gloves</td><td>Microflex</td><td>SU-690</td><td></td></tr><tr><td>600 ml beaker</td><td>Duran</td><td>2110648</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Delta</td><td>DC300H</td><td></td></tr><tr><td>Hygrometer</td><td>Wisewind</td><td>5330</td><td></td></tr><tr><td>Nitrogen gas flowmeter</td><td>Dwyer</td><td>RMA-6-SSV</td><td></td></tr><tr><td>K-type thermocouples</td><td>Digitron</td><td>311-1670</td><td></td></tr><tr><td>Centrifuge</td><td>Select BioProducts</td><td>Force Mini&amp;nbsp;</td><td></td></tr><tr><td>Pipette</td><td>Rainin</td><td>pipet-lite XLS</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Temperature controllable drying chamber</td><td>this lab</td><td></td><td></td></tr><tr><td>Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td><td>this lab</td><td></td><td></td></tr><tr><td>MALDI-TOF stainless steel sample target</td><td>this lab</td><td></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）的离子图像显示了类似的趋势，因为这些sodiated麦芽三糖。成像MS的结果表明，较低的样品板的温度下制备的样品可显著引入分子和降低异质性。 图3示出了用于下5和25℃的样品板的温度下制备麦芽三糖和激肽片段（1-7）的统计分析的结果。对于每个样品，平均强度进行归一化。在用25℃的样品板温度sodiated麦芽三糖的情况下，在中心的信号强度比为5℃下的样品板温度低得多。质子缓激肽片段（1-7）的结果也显示出较小的变化减小从25样品板温度至5℃时。 <img alt="图1" src="/files/ftp_upload/54409/54409fig1.jpg" /> 图1:样品干燥系统的图片。干燥室由丙烯酸制成。该室通入室温的氮气，以保持低的相对湿度条件。装备有编程恒温循环器的铜基块用于调节的不锈钢样品板的温度。温度计监测空气，铜基块，和样品板。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54409/54409fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54409/54409fig2.jpg" /> 图2:降低样品板的温度使得更好的信号的均匀性的明场图像（上部的图像）以及麦芽三（a）和激肽片段（1-7）的MALDI图像（低级图像）（b）与THAP制备在不同的样品板的温度。该MALDI<html>分别从总频谱，:和质子缓激肽片段（1-7）（757 M / Z）:图像通过提取sodiated麦芽三糖（527米/ Z）获得。离子图像的像素大小为35微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54409/54409fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54409/54409fig3.jpg" /> 图3: 信号变化减少作为样品板温度下干燥过程中降低的MALDI图像与麦芽三糖（a）和（b）根据不同的样品板的温度与THAP制备缓激肽片段（1-7）中获得。红色和蓝色的数据表明，在25和5℃的样品板的温度制备的样品，分别为。G"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品板温度（℃） </td><td> 样品 </td><td> 空气温度（℃） </td><td> 相对湿度（RH％） </td><td> 干燥时间（秒） </td></tr><tr><td rowspan="2">五</td><td>与THAP麦芽三糖</td><td rowspan="2"> 20±3 </td><td rowspan="4"> &lt;25 </td><td rowspan="2"> 800 - 1000 </td></tr><tr><td>缓激肽片段（1-7）与THAP </td></tr><tr><td rowspan="2"> 25 </td><td>与THAP麦芽三糖</td><td rowspan="2"> 25±3 </td><td rowspan="2"> 100 - 150 </td></tr><tr><td>缓激肽片段（1-7）与THAP </td></tr></tbody></table>表1: 实验参数和在不同的样品板的温度的干燥条件。

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

Nanyang Technological University

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

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

科研

化学

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 ...

生物学

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工程

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 ...

生物化学

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

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, ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...