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>
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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><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>2,5-dihydroxybenzoic acid (DHB)</td><td>Alfa Aesar</td><td>A11459</td><td></td></tr><tr><td>sialyl-lewis A (SLe&lt;sup&gt;A&lt;/sup&gt;)</td><td>Sigma-Aldrich</td><td>S1782</td><td></td></tr><tr><td>Maltoheptaose</td><td>Sigma-Aldrich</td><td>M7753</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>Vortex mixer</td><td>Scientific Industries</td><td>&amp;nbsp;SI-0236</td><td></td></tr><tr><td>Mini 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>Ultraflex II TOF/TOF mass spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>MTP 384 target plate polished steel BC</td><td>Bruker Daltonics</td><td>8280781</td><td></td></tr><tr><td>Flexcontrol Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Control software</td></tr><tr><td>Fleximaging Version 2.1</td><td>Bruker Daltonics</td><td></td><td>Imaging software</td></tr><tr><td>Flexanalysis Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Analysis software</td></tr></tbody></table>

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

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.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

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 ...

科研

生物化学

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 ...

化学

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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 ...

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 ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

使用循环离子淌度光谱仪进行串联离子淌度实验

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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 ...