Sabine Guenther

Histology by mass spectrometry: label-free tissue characterization obtained from high-accuracy bioanalytical imaging.

Histological examination of biological and medical specimens has gained its universality and undisputed significance through distinct staining techniques and microscopical evaluation. Discrimination of tissue types after specific staining or labeling is an essential prerequisite for histopathological investigation, for example in accurate diagnosis of cancer. Histochemical staining techniques can only be used in a targeted manner for known compounds, and only a limited number of such targets can be visualized from a given sample at the same time. Another limitation of classical histology lies in the fact that a considerable amount of experience is required and that even well-trained pathologists often interpret histologically stained sections differently. Mass spectrometry (MS), on the other hand, offers complex but objective and reproducible information on biological material. Imaging of biological samples by MS gained interest after development of matrix-assisted laser desorption/ionization (MALDI) as a method to desorb and ionize biomolecules, such as peptides, proteins, glycans, or lipids, with a limit of detection in the attomole range. The first proof-of-principle of imaging by MALDI was presented in 1994, and was followed by numerous applications during the last decade. An extensive overview of instrumental developments and methodological approaches in MS imaging has been published recently. MS imaging allows the distribution of analytes to be investigated and displayed across a sample in a semi-quantitative manner and without the need to predefine or label selected substances prior to analysis. MALDI imaging is typically used with spatial resolutions of between 50 and 200 mm. Increasing the resolution into the lowmicrometer range has been demonstrated, but requires a very low limit of detection of the employed mass spectrometer, as the available amount of material per imaged spot is reduced quadratically with reduction of the spot diameter. Identification of molecules during MS imaging experiments is often limited if mass spectrometers with a rather low mass resolving power and accuracy are used. Additional offline bulk analyses of tissue material are typically used to back up imaging results. Imaging selectivity, that is, mass bin width for allocation to image signals, is typically set to onemass unit. Employing MS imaging for obtaining valid histological information requires a number of improvements: 1. The usable spatial resolution has to be high enough to resolve cellular features. 2. Analytical sensitivity has to be high enough to visualize the majority of interesting substances in high-lateralresolution experiments. 3. Mass resolving power and mass accuracy have to be as high as possible when complex biological samples are under investigation. To unequivocally assign a mass signal to an image and to identify substances by accurate mass, signals have to be stable and correct in detected mass values; that is, mass accuracy should be in the low-ppm range. 4. Image assignment to mass signals has to be both highly selective and flexible. To distinguish neighboring mass signals in biological tissue samples, the coding mass bin width must typically be smaller than 0.1 mass units. 5. To clearly identify imaged substances in complex samples, MS data from fragmentation of precursor ions has to be obtainable directly from individual imaged sample spots. 6. Ambient pressure conditions are often necessary, rather than high-vacuum conditions, for example when working under physiological conditions, imaging volatile substances such as drug metabolites, or using volatile matrices. 7. Sample handling and preparation have to be fast and robust. 8. Results have to be achievable in a reasonable timeframe.

Single Cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100,000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.

A high-resolution scanning microprobe matrix-assisted laser desorption/ionization ion source for imaging analysis on an ion trap/Fourier transform ion cyclotron resonance mass spectrometer

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.

High‐resolution matrix‐assisted laser desorption/ionization imaging of tryptic peptides from tissue

RATIONALE The analysis of proteins by mass spectrometry imaging is an important biomedical application as spatial distributions can be used to identify markers for pathological processes. The direct detection and identification of proteins on tissue can be hindered by a number of factors including limited mass range and fragmentation efficiency as well as incompatibility with formalin-fixed samples. METHODS To overcome some of these limitations, on-tissue digestion of proteins was followed by detection of the resulting peptides. Trypsin was applied by a spraying device. Matrix-assisted laser desorption/ionization (MALDI) imaging experiments were performed with a home-built atmospheric-pressure imaging source attached to a LTQ Orbitrap mass spectrometer. The mass accuracy under imaging conditions was better than 3 ppm RMS. This allowed for confident identification of tryptic peptides by comparison with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) measurements of an adjacent mouse brain section. RESULTS A spatial resolution of 50 µm was obtained for tryptic peptides on tissue. Several tryptic peptides of myelin showed matching spatial distributions, and numerous tryptic peptides of other proteins were identified. MS images were generated with a bin size (mass range used for image generation) of Δm/z = 0.01 u. Examples demonstrate that MS images with lower selectivity can result in misleading information about the spatial distribution of tryptic peptides. CONCLUSIONS The presented method combines a significantly improved spatial resolution for tryptic peptides with low-ppm mass accuracy in a single experiment and thus provides highly reliable and specific information.

Mirion—A Software Package for Automatic Processing of Mass Spectrometric Images

AbstractMass spectrometric imaging (MSI) techniques are of growing interest for the Life Sciences. In recent years, the development of new instruments employing ion sources that are tailored for spatial scanning allowed the acquisition of large data sets. A subsequent data processing, however, is still a bottleneck in the analytical process, as a manual data interpretation is impossible within a reasonable time frame. The transformation of mass spectrometric data into spatial distribution images of detected compounds turned out to be the most appropriate method to visualize the results of such scans, as humans are able to interpret images faster and easier than plain numbers. Image generation, thus, is a time-consuming and complex yet very efficient task. The free software package “Mirion,” presented in this paper, allows the handling and analysis of data sets acquired by mass spectrometry imaging. Mirion can be used for image processing of MSI data obtained from many different sources, as it uses the HUPO-PSI-based standard data format imzML, which is implemented in the proprietary software of most of the mass spectrometer companies. Different graphical representations of the recorded data are available. Furthermore, automatic calculation and overlay of mass spectrometric images promotes direct comparison of different analytes for data evaluation. The program also includes tools for image processing and image analysis. Figureᅟ

A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods

RATIONALE An ideal method for bioanalytical applications would deliver spatially resolved quantitative information in real time and without sample preparation. In reality these requirements can typically not be met by a single analytical technique. Therefore, we combine different mass spectrometry approaches: chromatographic separation, ambient ionization and imaging techniques, in order to obtain comprehensive information about metabolites in complex biological samples. METHODS Samples were analyzed by laser desorption followed by electrospray ionization (LD-ESI) as an ambient ionization technique, by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging for spatial distribution analysis and by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) for quantitation and validation of compound identification. All MS data were acquired with high mass resolution and accurate mass (using orbital trapping and ion cyclotron resonance mass spectrometers). Grape berries were analyzed and evaluated in detail, whereas wheat seeds and mouse brain tissue were analyzed in proof-of-concept experiments. RESULTS In situ measurements by LD-ESI without any sample preparation allowed for fast screening of plant metabolites on the grape surface. MALDI imaging of grape cross sections at 20 µm pixel size revealed the detailed distribution of metabolites which were in accordance with their biological function. HPLC/ESI-MS was used to quantify 13 anthocyanin species as well as to separate and identify isomeric compounds. A total of 41 metabolites (amino acids, carbohydrates, anthocyanins) were identified with all three approaches. Mass accuracy for all MS measurements was better than 2 ppm (root mean square error). CONCLUSIONS The combined approach provides fast screening capabilities, spatial distribution information and the possibility to quantify metabolites. Accurate mass measurements proved to be critical in order to reliably combine data from different MS techniques. Initial results on the mycotoxin deoxynivalenol (DON) in wheat seed and phospholipids in mouse brain as a model for mammalian tissue indicate a broad applicability of the presented workflow.

Electrospray Post-Ionization Mass Spectrometry of Electrosurgical Aerosols

The feasibility of electrospray (ES) ionization of aerosols generated by electrosurgical disintegration methods was investigated. Although electrosurgery itself was demonstrated to produce gaseous ions, post-ionization methods were implemented to enhance the ion yield, especially in those cases when the ion current produced by the applied electrosurgical method is not sufficient for MS analysis. Post-ionization was implemented by mounting an ES emitter onto a Venturi pump, which is used for ion transfer. The effect of various parameters including geometry, high voltage setting, flow parameters, and solvent composition was investigated in detail. Experimental setups were optimized accordingly. ES post-ionization was found to yield spectra similar to those obtained by the REIMS technique, featuring predominantly lipid-type species. Signal enhancement was 20- to 50-fold compared with electrosurgical disintegration in positive mode, while no improvement was observed in negative mode. ES post-ionization was also demonstrated to allow the detection of non-lipid type species in the electrosurgical aerosol, including drug molecules. Since the tissue specificity of the MS data was preserved in the ES post-ionization setup, feasibility of tissue identification was demonstrated using different electrosurgical methods.