作者没有确认。

1. 样品板预适应
<ol><li>
样品板的清洗
	<ol>
<li>在清洗过程中, 应佩戴丁腈橡胶手套, 以避免样品板的污染。</li>
		<li>用100.0 毫升洗涤剂溶液 (1.0 毫克/毫升) 洗手样品板。</li>
		<li>用蒸馏-去离子水 (DDW) 洗手样品板。</li>
		<li>用30.0 毫升的甲醇冲洗样品板表面。</li>
		<li>把样品板放在600毫升烧杯中, 装满 DDW, 直到样品板完全浸入水中。</li>
		<li>把烧杯放在超声波浴中 (见 材料表), 油脂实验15分钟 (200 W, 40 赫) 的样品板。</li>
		<li>取出样品板, 用加压氮气吹掉水滴。</li>
		<li>在样品板上存入0.2 µL 甲醇, 以检查甲醇是否扩散到其他点。 注:如果甲醇与其他点合并, 重复步骤 1.1. 3-1. 1.5;如果没有, 请继续下一步。</li>
	</ol>
</li>
	<li>
干燥室温度的调节
	<ol>
<li>在稳定的条件下使用干燥室来干水滴, 如前所述11、12、13。特别是, 使用 2.1-2.5 的步骤中描述的详细过程, Y. m。等等。201612。简要:<ol>
<li>用室温氮气在恒定流速下清洗干燥室, 保持低相对湿度环境。</li>
			<li>保持样品板温度恒定在规则-(25 °c) 或快速干燥条件 (50 °c), 由温度控制铜块在烘干室调控。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
基质和分析物溶液的制备
	<ol>
<li>
基质溶液的制备
		<ol>
<li>溶解 DHB 在50% 乙腈 (ACN): 50% DDW 准备0.1 米溶液。</li>
		</ol>
</li>
		<li>
糖类分析剂的制备
		<ol>
<li>在 DDW 中溶解 SLea , 准备 10-4米溶液。</li>
			<li>在 DDW 溶解氢, 准备 10-4米溶液。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 样品沉积和再结晶
注:本文介绍了分析小样本和常规量样品的优化过程。在沉淀溶液之前, 确保样品板温度在所需温度下稳定下来。如果样品在一个大面积上扩散到其他样品点, 在再结晶过程中, 准备一个新的样品或重复步骤1.1。
<ol><li>
对样品的少量 (0.1 µL) 的分析 注意:为尽量减少采样和时间消耗, 已经制定了以下步骤。它适用于数量有限或快速 IMS 的实际样品定量分析。<ol>
<li>预混料0.25 µL DHB 溶液和0.25 µL 的 SLea或 MH 溶液在离心管。</li>
		<li>涡旋混合溶液使用涡旋混合器为 3 s。</li>
		<li>在微型离心机中, 旋转2秒 (2000 x g) 的混合溶液。</li>
		<li>使用吸管抽出0.1 µL 的预混溶液, 并立即将其存放在样品板上。 注:在沉积少量样品时,不要将预混溶液保存在吸管的尖端, 超过十年代。</li>
		<li>等待样品晾干。表 1列出了典型的干燥时间。</li>
		<li>用吸管将0.2 µL 的甲醇放在干燥的样品点上。样品马上就会弄湿并晾干。 注:确保沉积过程在3秒内完成, 以避免甲醇的显著蒸发损失。</li>
		<li>用显微镜检查样品。如果晶体形貌不像预期的那样 (见图 1所示的结果), 重复步骤 2.1. 1-2. 1.6 准备新样品。</li>
		<li>戴上丁腈橡胶手套, 小心从烘干室取出样品板。</li>
	</ol>
</li>
	<li>
对样品的常规量 (1 µL) 进行分析 注:下面的步骤是为了最大限度地利用 MALDI 样本量的碳水化合物样品的均匀性而制定的。该过程适用于常规和定量分析。再结晶过程将样品和基体均匀地重新分配到较大的区域。<ol>
<li>预混料2.5 µL DHB 溶液和2.5 µL SLea或 MH 溶液在离心管内。</li>
		<li>用涡流混合器对 5 s 的预混溶液进行涡旋。</li>
		<li>在微型离心机中, 旋转2秒 (2000 x g) 的混合溶液。</li>
		<li>使用吸管抽出1.0 µL 的预混溶液, 并立即将其存放在样品板上。 注:在沉积样品后不要再使用剩余的预混溶液。</li>
		<li>等待样品晾干。表 1列出了典型的干燥时间。</li>
		<li>用吸管将1.5 µL 的甲醇放在干燥的样品点上。样品马上就会弄湿并晾干。 注:在高试样温度 (50 °c) 的情况下, 应在5秒内进行再结晶步骤, 以最大限度地减少甲醇在吸管尖端的蒸发。</li>
		<li>用显微镜检查样品。如果晶体形貌不像预期的那样 (见图 1所示的结果), 重复步骤 2.2. 1-2. 2.6 准备新样品。</li>
		<li>戴上丁腈橡胶手套, 小心从烘干室取出样品板。</li>
	</ol>
</li>
</ol>3. 质谱数据的采集与分析
注:分析是使用一个商用飞行时间质谱仪 (材料表), 配备了 MALDI 离子源。该仪器由特定的控制软件 (材料表) 操作, 具有预优化的提取延时和激光能量。光谱以线性模式记录, 其质量范围为 m/z = 0-1500。样品板电位为±25凯文, 每种光谱平均10个激光射击。用户应使用兼容的软件进行仪器优化和样本分析, 并遵循仪表制造商的说明。
<ol><li>打开仪表控制软件 (见材料表)。</li>
	<li>将样品板插入质谱仪中。</li>
	<li>在软件中选择预优化的数据采集方法。</li>
	<li>使用成像软件为 IMS 注册整个采样区域 (见材料表)。 注:如果不执行 IMS, 请跳过此步骤。</li>
	<li>在控制软件的批处理模式下开始数据采集。</li>
	<li>在数据采集完成后, 用成像软件绘制出离子图像。</li>
	<li>如果没有离子图像记录数据, 则使用分析软件 (参见材料表) 分析质量谱。</li>
</ol>

<ol>
	<li>Holme, D. J., Peck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analytical Biochemistry. , (1998).</li><li>Costello, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time,+life+...+and+mass+spectrometry+-+New+techniques+to+address+biological+questions.">Time, life ... and mass spectrometry - New techniques to address biological questions.</a> Biophysical Chemistry. 68 (1-3), 173-188 (1997).</li><li>Caroff, M., Karibian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+lipopolysaccharides.">Structure of bacterial lipopolysaccharides.</a> Carbohydrate Research. 338 (23), 2431-2447 (2003).</li><li>Marvin, L. F., Roberts, M. A., Fay, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+in+clinical+chemistry.">Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in clinical chemistry.</a> Clinica Chimica Acta. 337 (1), 11-21 (2003).</li><li>Harvey, D. 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作者没有什么可透露的。

样本异质性是 MALDI 中最常用的样品制备方法, 但合成的晶体是高度异构的。此类样本显示了低投篮和采样采样信号重现性。因此, 在数据采集过程中寻找样本区域中的 "甜点" 是 MALDI 实验中的一个常见过程。这种非均匀样本在常规分析中不适合量化。
在目前的研究中, MALDI 样品形态学通过再结晶优化。再结晶对碳水化合物信号强度和数据稳定性的改善归因于碳水化合物与基质?...

碳水化合物分析是一个重要而具有挑战性的课题。碳水化合物和他们的衍生物在活有机体扮演重要角色1,2,3。这些分子结构复杂, 易于分解。由于分离和检测方面的困难, 其中许多不能清楚地描绘出来。虽然基质辅助激光解吸/电离 (MALDI) 质谱 (MS) 已被应用于分析范围广泛的生物分子, 由于其灵敏度和可理解的结果4, 分析碳水化合物使用 MALDI-MS 继续由于这种分子的电离效率低5, 这是一个主要的挑战。化学衍生是提高碳水化合物6、7的电离效率的常用方法, 但这种过程是时间和样本消耗。此外, 衍生物碳水化合物的电离效率仍然低于蛋白质。因此, 在不需要复杂程序的情况下, 改进 MALDI 中碳水化合物信号的方法是必要的。
MALDI-MS 在定量分析中的应用是另一个具有挑战性的课题。MALDI 的一个主要问题是它的灵敏度和数据重现性主要依赖于样品制备协议和实验参数。在许多情况下, 由于异质样品形貌和分析物分布, MALDI 的定量分析是不可靠的。一个众所周知的例子是用 25-苯甲酸酸 (DHB) MALDI 基质制备的样品。当 DHB 在周围环境下慢慢结晶时, 分析物被纳入基体晶体的程度是不可预知的, 因为结果样品显示不规则的形貌。这种样品通常由大针状和细晶体组成。当 DHB 使用挥发性溶剂和/或加热样品板制备时, 快速干燥过程会产生更均匀的细晶, 并取得更好的定量结果8、9、10。这种技术被称为 MALDI 样品的 "再结晶"。这种改进归因于在快速结晶过程中更好地将分析物纳入精细基体晶体中。我们还表明, 调整样品制备环境减少了碳水化合物信号的异质性和改进的定量结果11,12。这些研究结果表明, 样本形态学是确定碳水化合物信号质量的关键因素。为了制定日常分析的一般策略, 需要有一个有效的样本改革方法来提高碳水化合物的灵敏度。
我们有系统地研究了 MALDI-MS 样品形态学和碳水化合物敏感性的相关性, 在最近的报告13。利用几个重要的碳水化合物和矩阵得到的结果表明, 重结晶干 MALDI 样品能较好地实现最佳信号增强。用甲醇 (甲醇) 快速再结晶法对用常规干液滴 (DD) 方法制备的试样形貌进行了改造。详细的样品准备协议在这里展示。该协议包括样品板预处理、样品沉积和再结晶、质谱分析等三主要步骤。所使用的碳水化合物包括唾液酸化酶 (SLea) 和 maltoheptaose (MH)。DHB 用作模型矩阵。结果表明, 再结晶后碳水化合物信号强度和空间分布明显改善。这种方法可应用于其他常用基质的样品, 包括24、6-trihydroxyacetophenone (行动计划推行) 和αcyano-4-hydroxycinnamic 酸。这种方法是一种一般的方法, 可以很容易地集成在实验室例行的碳水化合物分析。

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Reagent</td>
			<td style="width:157px;"></td>
			<td style="width:137px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Detergent powder</td>
			<td style="width:157px;">Alconox</td>
			<td style="width:137px;">242985</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Methanol</td>
			<td style="width:157px;">Merck</td>
			<td style="width:137px;">106009</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Acetonitrile</td>
			<td style="width:157px;">Merck</td>
			<td style="width:137px;">100003</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">2,5-dihydroxybenzoic acid (DHB)</td>
			<td style="width:157px;">Alfa Aesar</td>
			<td style="width:137px;">A11459</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">sialyl-lewis A (SLeA)</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:137px;">S1782</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Maltoheptaose</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:137px;">M7753</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Pipette tips</td>
			<td style="width:157px;">Mettler Toledo</td>
			<td style="width:137px;">17005091</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Microcentrifuge tube</td>
			<td style="width:157px;">Axygen</td>
			<td style="width:137px;">MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Equipment</td>
			<td style="width:157px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Milli-Q water purification system</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:137px;">ZMQS6VFT1</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Powder-free nitrile gloves</td>
			<td style="width:157px;">Microflex</td>
			<td style="width:137px;">SU-690</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">600 mL beaker</td>
			<td style="width:157px;">Duran</td>
			<td style="width:137px;">2110648</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Ultrasonic cleaner</td>
			<td style="width:157px;">Delta</td>
			<td style="width:137px;">DC300H</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Hygrometer</td>
			<td style="width:157px;">Wisewind</td>
			<td style="width:137px;">5330</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Nitrogen gas flowmeter</td>
			<td style="width:157px;">Dwyer</td>
			<td style="width:137px;">RMA-6-SSV</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:332px;">K-type thermocouples</td>
			<td style="width:157px;">Digitron</td>
			<td style="width:137px;">311-1670</td>
			<td></td>
		</tr>
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			<td height="23" style="height:23px;width:332px;">Vortex mixer</td>
			<td style="width:157px;">Scientific Industries</td>
			<td style="width:137px;">&#160;SI-0236</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:332px;">Mini centrifuge</td>
			<td style="width:157px;">Select BioProducts</td>
			<td style="width:137px;">Force Mini&#160;</td>
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			<td height="23" style="height:23px;width:332px;">Pipette</td>
			<td style="width:157px;">Rainin</td>
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			<td height="23" style="height:23px;width:332px;">Temperature controllable drying chamber</td>
			<td style="width:157px;">This lab</td>
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			<td height="23" style="height:23px;width:332px;">Ultraflex II TOF/TOF mass spectrometer</td>
			<td style="width:157px;">Bruker Daltonics</td>
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			<td height="23" style="height:23px;width:332px;">MTP 384 target plate polished steel BC</td>
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			<td height="23" style="height:23px;width:332px;">Flexcontrol Version 3.4</td>
			<td style="width:157px;">Bruker Daltonics</td>
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			<td>Control software</td>
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			<td height="23" style="height:23px;width:332px;">Fleximaging Version 2.1</td>
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			<td>Analysis software</td>
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an efficient sample preparation method to enhance carbohydrate ion signals in matrix-assisted laser desorption/ionization mass spectrometry

样品制备是质谱 (MS) 分析碳水化合物的关键过程。虽然基质辅助激光解吸/电离 (MALDI) MS 是碳水化合物分析中的选择方法, 但碳水化合物样品的不良离子信号和数据重现性仍然是严重的问题。对于碳水化合物的定量分析, 必须有一个有效的分析协议, 提供卓越的数据质量。该视频演示了用于提高信号强度和减少 MALDI 中碳水化合物数据变化的样本准备协议。在样品滴的干燥和结晶后, 在质谱分析前, 甲醇对晶体形态进行了改造。用 MALDI 成像质谱 (IMS) 检测碳水化合物信号的增强。实验结果表明, 晶体的改造调整了晶体结构, 重新分配了碳水化合物的分析。与常规 MALDI 中的干液滴制备方法相比, 甲醇转化碳水化合物晶体形貌明显改善了信号强度、离子图像分布和数据稳定性。由于此处演示的协议不涉及样本组成的变化, 它们通常适用于各种碳水化合物和矩阵。

图 1显示了用 DD 和再结晶方法制备的 SLeA混合 DHB 的代表性 SEM 图像。DD 法制备的典型 DHB 形态为边缘处的大针状晶体和样品点中心的精细晶体结构。这种针状晶体的典型长度为100µm。经甲醇再结晶后, 试样的面积较大, 均匀地覆盖着细小的鳞片状晶体。"鳞片" 晶体的长度约为20-50 µm. 再结晶样品提供比传统 DD 样品所生产的更大的有效表面积。</...

Watch this Scientific Journal Video about An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry at JoVE.com

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

本文演示了在样品制备过程中, 通过改造晶体结构来增强 MALDI 质谱中的碳水化合物离子信号的协议。

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) ...

an-efficient-sample-preparation-method-to-enhance-carbohydrate-ion

Research

JoVE Journal

Chemistry

7.0K 次观看.  Academia Sinica. 本文演示了在样品制备过程中, 通过改造晶体结构来增强 MALDI 质谱中的碳水化合物离子信号的协议。

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

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 ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

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 ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

利用飞行时二次离子质谱法成像金属颜料界面的腐蚀

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

聚类诱导解吸/电电质谱法对复杂分子及其在表面的反应分析

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial resolution (~1 &#181;m). Furthermore, it describes how to obtain realistic three-dimensional (3D) distributions of segregated impurities/dopants in solid state materials. Direct 3D depth profile reconstruction is often difficult to achieve due to SIMS-related measurement artifacts. Presented here is a method to identify and solve this challenge. Three major issues are discussed, including the i) nonuniformity of the detector being compensated by flat-field correction; ii) vacuum background contribution (parasitic oxygen counts from residual gases present in the analysis chamber) being estimated and subtracted; and iii)&#160;performance of all steps within a stable timespan of the primary ion source. Wet chemical etching is used to reveal the position and types of dislocation in a material, then the SIMS result is superimposed on images obtained via scanning electron microscopy (SEM). Thus, the position of agglomerated impurities can be related to the position of certain defects. The method is fast and does not require sophisticated sample preparation stage; however, it requires a high-quality, stable ion source, and the entire measurement must be performed quickly to avoid deterioration of the primary beam parameters.

The presented method describes how to identify and solve measurement artifacts related to secondary ion mass spectrometry as well as obtain realistic 3D distributions of impurities/dopants in solid state materials.

使用二次离子质谱法重建分离杂质的 3D 深度剖面

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.