全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）
Summary
A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.
Abstract
对于质谱（MS）的环境电离源已在过去的十年中很大兴趣的主题。基质辅助激光解吸电喷雾电离（MALDESI）是这样的方法，一个例子，其中基质辅助激光解吸/电离（MALDI）（ 例如，解吸的脉冲性质）和电喷雾电离（ESI）（ 例如，软电离的特征）组合。之一的MALDESI的主要优点是其固有的多功能性。在MALDESI实验中，紫外线（UV）或红外线（IR）激光可用于共振激发内源或外源矩阵。矩阵的选择不是分析物依赖性的，并且仅取决于用于激发的激光波长。在IR-MALDESI实验中，冰的薄层沉积在样品表面作为能量吸收基质上。对IR-MALDESI源几何已经使用的试验设计（DOE），用于液体样品的分析统计设计以及生物化学优化ogical组织标本。此外，一强大的红外MALDESI成像源已被开发出来，其中，可调谐的中红外激光器用计算机控制的XY平移阶段和高的分辨能力质谱仪同步。自定义的图形用户界面（GUI）允许激光的重复频率的用户选择，每像素镜头，样品台的步长，和解吸之间的延迟和扫描源的事件的数目。红外MALDESI已在多种应用中被使用，如生物组织切片的纤维和染料和MSI的取证分析。不同的分析物，从内源性代谢物的组织切片中的外源外源物的分布可以被测量，并使用这种技术进行定量。在这个手稿介绍的协议详细描述了全身组织切片的IR-MALDESI MSI主要步骤。
Introduction
在微探针模式质谱成像（MSI）通过在样品的表面上的离散位置的光束（激光或离子）涉及从表面样品的解吸。在每个栅格点，则产生的质谱和所获取的光谱，与从它们被收集的空间位置一起，可以用来同时映射样本内的许多分析物。此无标记耦合到灵敏度和质谱特异性成像的方式帮助微星成为质谱1,2-最快速发展的领域之一。
基质辅助激光解吸/电离（MALDI）是用于MSI分析中最常见的电离方法。然而，需要一种有机基质和MALDI的真空要求姿势上再现性，样品通量，并且可以使用该方法分析的样品的类型显著局限性。许多大气压力（AP）的IOnization方法已被开发，近年来，以规避这些限制3。这些环境电离方法允许生物样品的分析中是更接近自然状态，并简化分析前样品制备步骤的环境。基质辅助激光解吸电喷雾电离（MALDESI）是这样一种电离法4,5的一个例子。
在IR-MALDESI实验中，冰的薄层沉积在组织表面作为能量吸收基质上。中红外激光脉冲由冰基质吸收，并促进中性材料的解吸从由共振激励OH表面伸展的水模式。解吸的中立分区为正交电的带电滴，并且后电离在ESI状方式4-6。外源性冰矩阵的加法优于在组织上的内源性水单靠，因为它有助于交流算用于在不同组织区室含水量的变化，并且已显示通过〜15倍的组织成像实验7,8，以提高解吸6和提高离子丰度。
在这项工作中，我们采用了IR-MALDESI MSI引发的新生小鼠全身不同器官的代谢产物的分布。对IR-MALDESI源的可调节的参数的概况给出，并且对组织切片的成功的成像必要的步骤被证明。
Protocol
Representative Results
Discussion
上述协议描述为执行IR-MALDESI MSI实验的关键步骤。矩阵应用程序（第3节）大约需要20分钟，这是通过升华或类似的用于MALDI微星实验的典型矩阵应用过程使用机器人喷雾器喷涂。此外，红外MALDESI不依赖于分析物的分配到基质晶体6和冰基质可普遍用于所有分析物，无论它们的质量，大小，或化学性质的。此外，利用冰的消除了在典型的MALDI实验23观察到较低的m / z范围的矩阵相…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.
Materials
| IR-MALDESI Source | Custom-made | N/A | Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development. |
| Q Exactive Plus | Thermo Scientific | Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer | |
| Water, HPLC Grade | Burdick & Jackson | AH365-4 | |
| Methanol, HPLC Grade | Burdick & Jackson | AH230-4 | |
| Formic Acid | Sigma Aldrich | 56302 | |
| Tunable mid-IR Laser | Opotek Inc. | IR Opolette | Tunable 2700-3100 nm IR OPO laser |
| Nitrogen Gas | Arc3 Gases | AG S-NI300-5.0 | Grade 5.0 high purity nitrogen gas cylinder (300) |
| Cryostat | Leica Biosystems | CM 1950 | Cryomicrotome |
| High Profile Microtome Blades | Leica Biosystems | 3802123 | Leica DB80HS |
| Mounting Medium (OCT) | Leica Biosystems | 3801480 | Surgipath FSC 22 mounting medium |
| Cryostat Specimen Disc | Leica Biosystems | 14047740045 | 40 mm diameter |
| Glass Microscope Slides | VWR | 48312-003 | Frosted, selected, pre-cleaned |
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