Dieter Staab

MALDI mass spectrometric imaging of biological tissue sections

In biomedical research, the discovery of new biomarkers and new drugs demands analytical techniques with high sensitivity together with increased throughput. The possibility to localize or to follow changes in organisms at the molecular level by imaging component distributions of specific tissues, is of prime importance to unravel biochemical pathways and develop new treatments and drugs. Established molecular imaging techniques such as MRI and PET are already widely used, however their need for molecular probes to report the presence of the analytes of interest precludes the simultaneous exploration of different biomolecules. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) takes full advantage of the high sensitivity of mass spectrometry instrumentation but also of the ability of the latter to simultaneously detect a wide range of compounds, almost regardless from their nature and mass. To perform MALDI MSI, sections of biological tissues are introduced in an MALDI MS instrument, where the UV pulsed laser of the MALDI source is used to raster over a selected area while acquiring mass spectra of the ablated ions at every image point. From this array of spectra, hundreds of analyte-specific images can be generated based on the selected masses. MALDI MSI can be used to track biomarkers such as peptides or proteins but also to map drug/tissue interactions. In this paper, an overview of the possibilities of MSI will be given. As an example, MSI on brain tissue sections for the study of Alzheimers disease (AD) will be shown. Mapping of amyloid peptides as a new approach for drug lead optimization will be presented. Target identification thanks to MSI will be introduced and the last part will be dedicated to the molecular scanner approach, which gives access to high-mass range by combining tissue blotting and digestion in a one-step process.

Molecular imaging of amyloid β peptides in mouse brain sections using mass spectrometry

A method is presented for direct spatial analysis of amyloid beta peptides in biological tissue sections. The technique takes advantage of the very high sensitivity of matrix-assisted laser desorption/ionization mass spectrometry and is implemented on a commercial instrument with modifications to only a few components and the software. With this setup, hundreds of molecular images can be generated simultaneously and within just a few minutes. The current features are an instrumental resolution of 50 microm and a sensitivity down to the attomol range. This new technology is applied to the study of amyloid beta peptide distribution in brain sections from mice, showing features reminiscent of Alzheimers disease.

High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.

Dynamics of Aβ Turnover and Deposition in Different β-Amyloid Precursor Protein Transgenic Mouse Models Following γ-Secretase Inhibition

Human β-amyloid precursor protein (APP) transgenic mice are commonly used to test potential therapeutics for Alzheimers disease. We have characterized the dynamics of β-amyloid (Aβ) generation and deposition following γ-secretase inhibition with compound LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide]. Kinetic studies in preplaque mice distinguished a detergent-soluble Aβ pool in brain with rapid turnover (half-lives for Aβ40 and Aβ42 were 0.7 and 1.7 h) and a much more stable, less soluble pool. Aβ in cerebrospinal fluid (CSF) reflected the changes in the soluble brain Aβ pool, whereas plasma Aβ turned over more rapidly. In brain, APP C-terminal fragments (CTF) accumulated differentially. The half-lives for γ-secretase degradation were estimated as 0.4 and 0.1 h for C99 and C83, respectively. Three different APP transgenic lines responded very similarly to γ-secretase inhibition regardless of the familial Alzheimers disease mutations in APP. Amyloid deposition started with Aβ42, whereas Aβ38 and Aβ40 continued to turn over. Chronic γ-secretase inhibition lowered amyloid plaque formation to a different degree in different brain regions of the same mice. The extent was inversely related to the initial amyloid load in the region analyzed. No evidence for plaque removal below baseline was obtained. γ-Secretase inhibition led to a redistribution of intracellular Aβ and an elevation of CTFs in neuronal fibers. In CSF, Aβ showed a similar turnover as in preplaque animals demonstrating its suitability as marker of newly generated, soluble Aβ in plaque-bearing brain. This study supports the use of APP transgenic mice as translational models to characterize Aβ-lowering therapeutics.

Transgenic Expression of Intraneuronal Aβ42 But Not Aβ40 Leads to Cellular Aβ Lesions, Degeneration, and Functional Impairment without Typical Alzheimer's Disease Pathology

An early role of amyloid-β peptide (Aβ) aggregation in Alzheimers disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of Aβ to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing Aβ1–40 (APP47) and Aβ1–42 (APP48) with a cleaved signal sequence to insert both peptides during synthesis into the endoplasmic reticulum. Although lower in transgene mRNA, APP48 mice reach a higher brain Aβ concentration. The reduced solubility and increased aggregation of Aβ1–42 may impair its degradation. APP48 mice develop intracellular Aβ lesions in dendrites and lysosomes. The hippocampal neuron number is reduced already at young age. The brain weight decreases during aging in conjunction with severe white matter atrophy. The mice show a motor impairment. Only very few Aβ1–40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of Aβ1–42 in APP48 mice does not lead to the AD-typical lesions, Aβ aggregates develop within cells accompanied by considerable neurodegeneration.

MALDI MS imaging of amyloid.

Label-free molecular imaging by mass spectrometry allows simultaneous mapping of multiple analytes in biological tissue sections. In this chapter, the application of this new technology to the detection Abeta peptides in mouse brain sections is discussed.

Applications of MALDI-MSI to Pharmaceutical Research

MALDI-MSI has been demonstrated to be a suitable technique in pharmaceutical research for providing information of the distribution of low molecular weight compounds such as drugs and their metabolites within whole-body tissue sections. Important ADME information can be determined by MALDI-MSI analysis of the distribution of drugs and metabolites in whole-body tissue sections taken from animals killed at a range of time points postdose. In this example we applied MALDI-MSI to the localization of a compound and its primary metabolite in whole-body mouse sections.

Reproducible Matrix Deposition for MALDI MSI Based on Open-Source Software and Hardware

AbstractThe new open-source software and hardware matrix deposition device named iMatrixSpray was optimized and specified for homogeneity, reproducibility, and sensitivity in MS imaging experiments. The results confirm the design claims, with the device delivering uniform coatings with a constant quality from experiment to experiment. The robustness in combination with the open design allows developing and sharing of matrix deposition and sample preparation protocols between labs. This tool therefore enables researchers to enter the field of MALDI MSI without previous experience in matrix coating. Graphical Abstractᅟ

iMatrixSpray: a free and open source sample preparation device for mass spectrometric imaging.

A device was built for matrix deposition in mass spectrometric imaging. This spray-type instrument requires no user interaction other than providing the spray solution and selecting the pre-defined or custom-built method. Robustness was achieved by utilizing a delta-robotics design in combination with a simple liquid system. All the information describing the systems is provided as open source and hardware and the design is therefore suitable for wide distribution and adaption by the scientific community.

Biophotonics Applied to Proteomics

Since the completion of the human genome sequencing, our understanding of gene and protein function and their involvement in physiopathological states has increased dramatically, partly due to technological developments in photonics. Photonics is a very active area where new developments occur on a weekly basis, while established tools are adapted to fulfill the needs of other disciplines like genomics and proteomics. Biophotonics emerged at the interface of photonics and biology as a very straightforward and efficient approach to observe and manipulate living systems. In this chapter, we review the current applications of photonics and imaging to proteomics from 2D gels analysis to molecular imaging.