Jonathan Stauber

Sample preparation issues for tissue imaging by imaging MS.

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.

On-Tissue Protein Identification and Imaging by MALDI-Ion Mobility Mass Spectrometry

MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues—formalin fixed paraffin embedded (FFPE) and frozen tissues—are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics “bottom-up” strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.

Quantitative mass spectrometry imaging of propranolol and olanzapine using tissue extinction calculation as normalization factor.

In order to quantify small molecules at the early stage of drug discovery, we developed a quantitation approach based on mass spectrometry imaging (MSI) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) without the use of a labeled compound. We describe a method intended to respond to the main challenges encountered in quantification through MALDI imaging dedicated to whole-body or single heterogeneous organ samples (brain, eye, liver). These include the high dependence of the detected signal on the matrix deposition, the MALDI ionization yield of specific target molecules, and lastly, the ion suppression effect on the tissue. To address these challenges, we based our approach on the use of a normalization factor called the TEC (Tissue Extinction Coefficient). This factor takes into account the ion suppression effect that is both tissue- and drug-specific. Through this protocol, the amount of drug per gram of tissue was determined, which in turn, was compared with other analytical techniques such as Liquid Chromatography-Mass spectrometry (LC-MS/MS).

Imaging mass spectrometry: Hype or hope?

Imaging mass spectrometry is currently receiving a significant amount of attention in the mass spectrometric community. It offers the potential of direct examination of biomolecular patterns from cells and tissue. This makes it a seemingly ideal tool for biomedical diagnostics and molecular histology. It is able to generate beautiful molecular images from a large variety of surfaces, ranging from cancer tissue sections to polished cross sections from old-master paintings. What are the parameters that define and control the implications, challenges, opportunities, and (im)possibilities associated with the application of imaging MS to biomedical tissue studies. Is this just another technological hype or does it really offer the hope to gain new insights in molecular processes in living tissue? In this critical insight this question is addressed through the discussion of a number of aspects of MS imaging technology and sample preparation that strongly determine the outcome of imaging MS experiments.

Perspectives for imaging mass spectrometry in the proteomics landscape

A number of techniques are used in the field of proteomics that can be combined to get the most molecular information from a specific biological sample, fluid or tissue. Imaging techniques are often used to obtain local information from tissue samples. However, imaging experiments are often staining experiments, which rely on specific or aspecific interactions between fluorescent markers and pre‐defined (families of) peptide or protein. Therefore, imaging is often used as a screening or validation tool for the local presence of proteins that have been identified by other means. Imaging mass spectrometry (IMS) combines the advantages of MS and microscopy in a single experiment. It is a technique that does not require any labeling of the analytes and provides a high multiplexing capability combined with the potential for analyte identification. It enables simultaneous detection of potentially all peptides and proteins present at a tissue surface and is used for the determination and identification of tissue‐specific disease markers. The workflows of IMS experiments closely resemble those of conventional proteomics. In this review, we describe IMS experiments step‐by‐step to position and evaluate the role of IMS in a comparative proteomics landscape. We illustrate in a concise review that IMS is a true discovery oriented tool for proteomics that seamlessly integrates in conventional proteomics workflows and can be perceived as either an alternative or complementary proteomics technique.

A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits

We investigated in a rabbit model, the eye distribution of topically instilled benzalkonium_(BAK) chloride a commonly used preservative in eye drops using mass spectrometry imaging. Three groups of three New Zealand rabbits each were used: a control one without instillation, one receiving 0.01%BAK twice a day for 5 months and one with 0.2%BAK one drop a day for 1 month. After sacrifice, eyes were embedded and frozen in tragacanth gum. Serial cryosections were alternately deposited on glass slides for histological (hematoxylin-eosin staining) and immunohistological controls (CD45, RLA-DR and vimentin for inflammatory cell infiltration as well as vimentin for Müller glial cell activation) and ITO or stainless steel plates for MSI experiments using Matrix-assisted laser desorption ionization time-of-flight. The MSI results were confirmed by a round-robin study on several adjacent sections conducted in two different laboratories using different sample preparation methods, mass spectrometers and data analysis softwares. BAK was shown to penetrate healthy eyes even after a short duration and was not only detected on the ocular surface structures, but also in deeper tissues, especially in sensitive areas involved in glaucoma pathophysiology, such as the trabecular meshwork and the optic nerve areas, as confirmed by images with histological stainings. CD45-, RLA-DR- and vimentin-positive cells increased in treated eyes. Vimentin was found only in the inner layer of retina in normal eyes and increased in all retinal layers in treated eyes, confirming an activation response to a cell stress. This ocular toxicological study confirms the presence of BAK preservative in ocular surface structures as well as in deeper structures involved in glaucoma disease. The inflammatory cell infiltration and Müller glial cell activation confirmed the deleterious effect of BAK. Although these results were obtained in animals, they highlight the importance of the safety-first principle for the treatment of glaucoma patients.

MALDI imaging techniques dedicated to drug-distribution studies

Previously, MS was often used to analyze the composition and structure of biological molecules present in solutions. Today, technology developments enable the application of MS for the analysis of localized biomolecules on biological tissue surfaces. This technique is called MS imaging. MALDI imaging MS is a technique whereby thousands of compounds present in a tissue section are detected simultaneously without labeling. Although initially used for the detection of biomolecules such as peptides and proteins, this technology is also used today for drug detection. These characteristics make MS imaging an ideal technology that is perfectly adapted for ADME (absorption, distribution, metabolism and excretion) studies. In fact, this technology facilitates the tracking of one or several administered drugs, as well as the metabolites that result from their assimilations. In this article, we will present the various possibilities that MALDI imaging MS approaches have to offer for the study of drugs and their metabolites using MS, MS/MS, FAST-SRM and MRM modes. In this context, we investigate two studies: the distribution of olanzapine in the kidney and the overall distribution of BDM31343 in mouse whole-body section.

Specific MALDI-MSI: TAG-MASS

MALDI imaging as a molecular mass spectrometry imaging technique (MSI) can provide accurate information about molecular composition on a surface. The last decade of MSI development has brought the technology to clinical and biomedical applications as a complementary technique of MRI and other molecular imaging. Then, this IMS technique is used for endogenous and exogenous molecule detection in pharmaceutical and biomedical fields. However, some limitations still exist due to physical and chemical aspects, and sensitivity of certain compounds is very low. Thus, we developed a multiplex technique for fast detection of different compound natures. The multiplex MALDI imaging technique uses a photocleavable group that can be detect easily by MALDI instrument. These techniques of targeted imaging using Tag-Mass molecules allow the multiplex detection of compounds like antibodies or oligonucleotides. Here, we describe how we used this technique to detect huge proteins and mRNA by MALDI imaging in rat brain and in a model for regeneration; the leech.

Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator

AMP-activated protein kinase (AMPK) is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0)) accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0) significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0) could be responsible for the enhanced ATP production via activation of the glycolytic pathway.

Label-free MS imaging from drug discovery to preclinical development

To fully understand the drug mechanism of action of new chemical entities, pharmacologists need to acquire confident and precise data in pharmacokinetics and in pharmacodynamics and build strong pharmacokinetic/pharmacodynamic relationships. Target engagement in evaluating new chemical entities provides the basis for treatment efficacy. Classical technologies are sometimes limited or inefficient to provide these precise data; however, label-free MS imaging technology is able to provide these molecular features, spatial distributions, quantification and metabolomics data. Important considerations for imaging biological sections are described. Various applications in pharmacology are presented across different therapeutic areas, where MS imaging answers crucial drug discovery and preclinical development needs.