Digital Imaging Mass Spectrometry

Methods to visualize the two-dimensional (2D) distribution of molecules by mass spectrometric imaging evolve rapidly and yield novel applications in biology, medicine, and material surface sciences. Most mass spectrometric imagers acquire high mass resolution spectra spot-by-spot and thereby scan the object’s surface. Thus, imaging is slow and image reconstruction remains cumbersome. Here we describe an imaging mass spectrometer that exploits the true imaging capabilities by ion optical means for the time of flight mass separation. The mass spectrometer is equipped with the ASIC Timepix chip as an array detector to acquire the position, mass, and intensity of ions that are imaged by matrix-assisted laser desorption/ionization (MALDI) directly from the target sample onto the detector. This imaging mass spectrometer has a spatial resolving power at the specimen of (84 ± 35) μm with a mass resolution of 45 and locates atoms or organic compounds on a surface area up to ~2 cm². Extended laser spots of ~5 mm² on structured specimens allows parallel imaging of selected masses. The digital imaging mass spectrometer proves high hit-multiplicity, straightforward image reconstruction, and potential for high-speed readout at 4 kHz or more. This device demonstrates a simple way of true image acquisition like a digital photographic camera. The technology may enable a fast analysis of biomolecular samples in near future.     

Identification of Biomarkers of Necrosis in Xenografts Using Imaging Mass Spectrometry

Xenografts are commonly used to test the effect of new drugs on human cancer. However, because of their heterogeneity, analysis of the results is often controversial. Part of the problem originates in the existence of tumor cells at different metabolic stages: from metastatic to necrotic cells, as it happens in real tumors. Imaging mass spectrometry is an excellent solution for the analysis of the results as it yields detailed information not only on the composition of the tissue but also on the distribution of the biomolecules within the tissue. Here, we use imaging mass spectrometry to determine the distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and their plasmanyl- and plasmenylether derivatives (PC-P/O and PE-P/O) in xenografts of five different tumor cell lines: A-549, NCI-H1975, BX-PC3, HT29, and U-87 MG. The results demonstrate that the necrotic areas showed a higher abundance of Na⁺ adducts and of PC-P/O species, whereas a large abundance of PE-P/O species was found in all the xenografts. Thus, the PC/PC-ether and Na⁺/K⁺ ratios may highlight the necrotic areas while an increase on the number of PE-ether species may be pointing to the existence of viable tumor tissues. Furthermore, the existence of important changes in the concentration of Na⁺ and K⁺ adducts between different tissues has to be taken into account while interpreting the imaging mass spectrometry results.

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Immuno-Desorption Electrospray Ionization Mass Spectrometry Imaging Identifies Functional Macromolecules by Using Microdroplet-Cleavable Mass Tags

We present immunoassay-based desorption electrospray ionization mass spectrometry imaging (immuno-DESI-MSI) to visualize functional macromolecules such as drug targets and cascade signaling factors. A set of boronic acid mass tags (BMTs) were synthesized to label antibodies as MSI probes. The boronic ester bond is employed to cross-link the BMT with the galactosamine-modified antibody. The BMT can be released from its tethered antibody by ultrafast cleavage of the boronic ester bond caused by the acidic condition of sprayed DESI microdroplets containing water. The fluorescent moiety enables the BMT to work in both optical and MS imaging modes. The positively charged quaternary ammonium group enhances the ionization efficiency. The introduction of the boron element also makes mass tags readily identified because of its unique isotope pattern. Immuno-DESI-MSI provides an appealing strategy to spatially map macromolecules beyond what can be observed by conventional DESI-MSI, provided antibodies are available to the targeted molecules of interest.     

Mass Recalibration of FT-ICR Mass Spectrometry Imaging Data Using the Average Frequency Shift of Ambient Ions

Achieving and maintaining high mass measurement accuracy (MMA) throughout a mass spectrometry imaging (MSI) experiment is vital to the identification of the observed ions. However, when using FTMS instruments, fluctuations in the total ion abundance at each pixel due to inherent biological variation in the tissue section can introduce space charge effects that systematically shift the observed mass. Herein we apply a recalibration based on the observed cyclotron frequency shift of ions found in the ambient laboratory environment, polydimethylcyclosiloxanes (PDMS). This calibration method is capable of achieving part per billion (ppb) mass accuracy with relatively high precision for an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI dataset. Comparisons with previously published mass calibration approaches are also presented.

基于非负分解方法的质谱成像数据特征提取

质谱成像技术能够在同一个实验里无需标记手段而获得样品表面的分子信息及其分布信息,是当前质谱分析的热点.其分析所得数据量大且复杂,使其特征难以提取.多元统计分析方法,特别是主成分分析法已应用于质谱成像数据的压缩和特征提取.然而由于主成分分析常产生负的数据结果,其意义难以解释且不易分解为单一的特征.本研究开发出一种基于非负分解的质谱成像数据提取方法,能够提取单一的分子特征及其在样品上的分布特征,并将多个单一的特征分布通过红、绿、蓝三色叠加显示,获得轮廓直观的综合特征分布.应用本方法对小鼠脑组织切片质谱成像数据进行分析,可直观分解出灰质区域、白质区域和背景区域,相对主成分分析方法更直观且易于解释.应用本方法对在同一个样品靶上的人膀胱癌变组织和其相邻非癌变组织切片质谱成像数据进行分析,癌变与非癌变组织间差异清晰直观.本研究设计的质谱成像软件可由http://www.msimaging.net获取.     

IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging of biological tissue sections using a layer of deposited ice as an energy-absorbing matrix was investigated. Dynamics of plume ablation were first explored using a nanosecond exposure shadowgraphy system designed to simultaneously collect pictures of the plume with a camera and collect the Fourier transform ion cyclotron resonance FT-ICR mass spectrum corresponding to that same ablation event. Ablation of fresh tissue analyzed with and without using ice as a matrix were compared using this technique. Effect of spot-to-spot distance, number of laser shots per pixel, and tissue condition (matrix) on ion abundance were also investigated for 50 μm-thick tissue sections. Finally, the statistical method called design of experiments was used to compare source parameters and determine the optimal conditions for IR-MALDESI of tissue sections using deposited ice as a matrix. With a better understanding of the fundamentals of ablation dynamics and a systematic approach to explore the experimental space, it was possible to improve ion abundance by nearly one order of magnitude.

Anatomical-Molecular Distribution of EphrinA1 in Infarcted Mouse Heart Using MALDI Mass Spectrometry Imaging

EphrinA1 is a tyrosine kinase receptor localized in the cellular membrane of healthy cardiomyocytes, the expression of which is lost upon myocardial infarction (MI). Intra-cardiac injection of the recombinant form of ephrinA1 (ephrinA1-Fc) at the time of ligation in mice has shown beneficial effects by reducing infarct size and myocardial necrosis post-MI. To date, immunohistochemistry and Western blotting comprise the only experimental approaches utilized to localize and quantify relative changes of ephrinA1 in sections and homogenates of whole left ventricle, respectively. Herein, we used matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) coupled with a time-of-flight mass spectrometer (MALDI/TOF MS) to identify intact as well as tryptic fragments of ephrinA1 in healthy controls and acutely infarcted murine hearts. The purpose of the present study was 3-fold: (1) to spatially resolve the molecular distribution of endogenous ephrinA1, (2) to determine the anatomical expression profile of endogenous ephrinA1 after acute MI, and (3) to identify molecular targets of ephrinA1-Fc action post-MI. The tryptic fragments detected were identified as the ephrinA1-isoform with 38% and 34% sequence coverage and Mascot scores of 25 for the control and MI hearts, respectively. By using MALDI-MSI, we have been able to simultaneously measure the distribution and spatial localization of ephrinA1, as well as additional cardiac proteins, thus offering valuable information for the elucidation of molecular partners, mediators, and targets of ephrinA1 action in cardiac muscle.

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二氢黄酮的电喷雾质谱研究

随着研究方法和技术的不断提高,人们发现生物类黄酮有很多新的种类和生理作用[1].中药苦参是豆科槐属植物苦参(Sophora Flavaescens Ait)的干燥根,含有丰富的黄酮类化合物,具有抗心率不齐等药理作用,特别是Kushenol A、Kurainone、Kuraridin为CAMP磷酸二酯酶的抑制剂[2].本文首次利用电喷雾多级串联质谱(ESI-MSn)的先进技术对苦参总黄酮中的2种二氢黄酮类化合物(kushenol A和nor-kurarinol)进行了多级串联质谱研究,得出了其在电喷雾条件下的碎片信息,为研究化合物在电喷雾条件下的裂解规律提供了一定的证据.     

Advanced Mass Calibration and Visualization for FT-ICR Mass Spectrometry Imaging

Mass spectrometry imaging by Fourier transform ion cyclotron resonance (FT-ICR) yields hundreds of unique peaks, many of which cannot be resolved by lower performance mass spectrometers. The high mass accuracy and high mass resolving power allow confident identification of small molecules and lipids directly from biological tissue sections. Here, calibration strategies for FT-ICR MS imaging were investigated. Sub-parts-per-million mass accuracy is demonstrated over an entire tissue section. Ion abundance fluctuations are corrected by addition of total and relative ion abundances for a root-mean-square error of 0.158?ppm on 16,764 peaks. A new approach for visualization of FT-ICR MS imaging data at high resolution is presented. The ??Mosaic Datacube?? provides a flexible means to visualize the entire mass range at a mass spectral bin width of 0.001?Da. The high resolution Mosaic Datacube resolves spectral features not visible at lower bin widths, while retaining the high mass accuracy from the calibration methods discussed.     

Preparative Mass Spectrometry Using a Rotating-Wall Mass Analyzer

The design of functional interfaces is central to both fundamental and applied research in materials science and energy technology. We introduce a new, broadly applicable technique for the precisely controlled high-throughput preparation of well-defined interfaces containing polyatomic species ranging from small ions to nanocrystals and large protein complexes. The mass-dispersive deposition of ions onto surfaces is achieved using a rotating-wall mass analyzer, a compact device which enables the separation of ions using low voltages and has a theoretically unlimited mass range. We demonstrate an efficient deposition of singly charged Au₁₄₄(SC₄H₉)₆₀ ions (33.7 kDa), which opens up exciting opportunities for the structural characterization of nanocrystals and their assemblies using transmission electron microscopy. Our approach also enables the high-throughput deposition of mass-selected ions from multicomponent mixtures, which is of interest to the controlled preparation of surface gradients and rapid screening of molecules in mixtures for a specific property.     

MALDI Mass Spectrometry Imaging of Neuronal Cell Cultures

Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI—at single cell spatial resolution—in both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched, the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation of a variety of cell cultures, including functioning neuronal networks.     

Microanalysis Using Secondary Ion Mass Spectrometry

Secondary ion mass spectrometry (SIMS) has inherent features of high sensitivity, great dynamic range, and capability to provide spatially resolved chemical information making it well suited for trace and microanalysis of diverse materials. The various SIMS methods used to derive the boron distribution in hepatoma cells, to investigate the intcr-iayer reactions in multi-layer ceramic structural materials, and to evaluate the effects of fabrication on microstructural and functional properties in semiconductor devices, are presented to illustrate possible roles of SIMS in microanalysis.     

Data Processing for 3D Mass Spectrometry Imaging

Data processing for three dimensional mass spectrometry (3D-MS) imaging was investigated, starting with a consideration of the challenges in its practical implementation using a series of sections of a tissue volume. The technical issues related to data reduction, 2D imaging data alignment, 3D visualization, and statistical data analysis were identified. Software solutions for these tasks were developed using functions in MATLAB. Peak detection and peak alignment were applied to reduce the data size, while retaining the mass accuracy. The main morphologic features of tissue sections were extracted using a classification method for data alignment. Data insertion was performed to construct a 3D data set with spectral information that can be used for generating 3D views and for data analysis. The imaging data previously obtained for a mouse brain using desorption electrospray ionization mass spectrometry (DESI-MS) imaging have been used to test and demonstrate the new methodology.     

Mass Determination of Entire Amyloid Fibrils by Using Mass Spectrometry

Amyloid fibrils are self‐assembled protein structures with important roles in biology (either pathogenic or physiological), and are attracting increasing interest in nanotechnology. However, because of their high aspect ratio and the presence of some polymorphism, that is, the possibility to adopt various structures, their characterization is challenging and basic information such as their mass is unknown. Here we show that charge‐detection mass spectrometry, recently developed for large self‐assembled systems such as viruses, provides such information in a straightforward manner.     

质谱分子成像技术与应用进展

该文总结了二次离子质谱、基质辅助激光解吸电离质谱和常压敞开式离子化质谱三大类型质谱分子成像（MSI）技术的概况、技术与方法及其应用新进展。MSI技术作为免标记、高覆盖、高灵敏、检测范围广的可视化分析手段,不局限于生物组织或细胞中某种特定分子的检测,可对已知和未知多种分子进行同时成像分析,获得不同分子的空间分布、相对含量及结构信息,实现其分子的定性、定量与定位分析;还可提供不同生理及病理过程中功能分子的动态时空变化信息等。因此,MSI技术成为质谱领域以及分析化学等领域的研究前沿与热点方向之一,并在化学、医学、生命科学、药学和环境科学等领域显示出重大应用前景。此外,MSI技术是单细胞可视化分析和空间分辨代谢组学的强有力分析手段,可从动物或器官组织的整体、微区、单细胞等不同空间尺度,获取具有空间分布特征、时空动态变化的功能分子全景轮廓信息等而备受关注。     

基于质谱成像和组学分析的环境毒理研究

生物体多器官的空间异质性导致环境污染物在生物体内的毒性分子机制错综复杂.基于传统化学和生物分析的环境毒理学研究,通常将研究对象看作“均一”整体,无法从空间上准确定位污染物及其代谢.以质谱成像和组学分析为基础的技术,同时对污染物、污染物代谢活化途径及其诱导的生物分子进行定性、定量和空间分析,从而确定污染物迁移、生物学效应...     

IR-MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution

High spatial resolution in mass spectrometry imaging (MSI) is crucial to understanding the biology dictated by molecular distributions in complex tissue systems. Here, we present MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) at 50 μm resolution. An adjustable iris, beam expander, and an aspherical focusing lens were used to reduce tissue ablation diameters for MSI at high resolution. The laser beam caustic was modeled using laser ablation paper to calculate relevant laser beam characteristics. The minimum laser spot diameter on the tissue was determined using tissue staining and microscopy. Finally, the newly constructed optical system was used to image hen ovarian tissue with and without oversampling, detailing tissue features at 50 μm resolution.

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质谱成像方法及其在临床研究中的应用

生物组织质谱成像技术(imaging mass spectrometry,IMS)是一种分子成像技术,它与荧光标记的激光共聚焦、整体动物放射自显影术和正电子发射断层扫描等其它分子成像不同,不再使成像局限于特异分子,而是面向组织中任何分子,不用荧光或放射性同位素标记,不需要进行蛋白质或多肽提取等复杂的样品前处理.