David Bonnel

MALDI Imaging Mass Spectrometry STATE OF THE ART TECHNOLOGY IN CLINICAL PROTEOMICS

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patients entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.

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).

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer

The large amount of data generated using matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) poses a challenge for data analysis. In fact, generally about 1.108–1.109 values (m/z, I) are stored after a single MALDI-MSI experiment. This imposes processing techniques using dedicated informatics tools to be used since manual data interpretation is excluded. This work proposes and summarizes an approach that utilizes a multivariable analysis of MSI data. The multivariate analysis, such as principal component analysis–symbolic discriminant analysis, can remove and highlight specific m/z from the spectra in a specific region of interest. This approach facilitates data processing and provides better reproducibility, and thus, broadband acquisition for MALDI-MSI should be considered an effective tool to highlight biomarkers of interest. Additionally, we demonstrate the importance of the hierarchical classification of biomarkers by analyzing studies of clusters obtained either from digested or undigested tissues and using bottom-up and in-source decay strategies for in-tissue protein identification. This provides the possibility for the rapid identification of specific markers from different histological samples and their direct localization in tissues. We present an example from a prostate cancer study using formalin-fixed paraffin-embedded tissue.

MITICS (MALDI Imaging Team Imaging Computing System): A new open source mass spectrometry imaging software

MITICS is a new software developed for MALDI imaging. We tried to render this software compatible with all types of instruments. MITICS is divided in two parts: MITICS control for data acquisition and MITICS Image for data processing and images reconstruction. MITICS control is available for Applied BioSystems MALDI-TOF instruments and MITICS Image for both Applied BioSystems and Bruker Daltonics ones. MITICS Control provides an interface to the user for setting the acquisition parameters for the imaging sequence, namely set instruments acquisition parameters, create the raster of acquisition and control post-acquisition data processing, and provide this settings to the automatic acquisition software of the MALDI instrument. MITICS Image ensures image reconstruction, files are first converted to XML files before being loaded in a database. In MITICS image we have chosen to implement different data representations and calculations for image reconstruction. MITICS Image uses three different representations that have shown to ease extraction of information from the whole data set. It also offers image reconstruction base either on the maximum peak intensity or the peak area. Image reconstruction is possible for single ions but also by summing signals of different ions. MITICS was validated on biological cases.

Localization of secondary metabolites in marine invertebrates: contribution of MALDI MSI for the study of saponins in Cuvierian tubules of H. forskali.

Background Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. Results These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. Conclusions All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates.

Ionic matrices pre-spotted matrix-assisted laser desorption/ionization plates for patient maker following in course of treatment, drug titration, and MALDI mass spectrometry imaging.

In the current study, we compared plastic matrix-assisted laser desorption/ionization (MALDI) plates pre-spotted with different solid ionic matrices. Data reflect that after 3 months of storage, the standards were oxidized in α-cyano-4-hydroxycinnamic acid (HCCA) whether or not in HCCA/3-acetylpyridine (3APY) and HCCA/aniline, and certain peptides, such as ubiquitin, were not detected using the HCCA matrix, whereas they were detected in pre-spotted ionic matrices. Application in peptidomics of these MALDI matrices pre-spotted plates (after 3 months of storage) with ovarian cyst fluid showed less intense signals with HCCA than with solid ionic matrices. We show that these pre-spotted ionic matrices plates can be used for relative drug quantification, high mass protein detection, and MALDI mass spectrometry imaging.

MALDI imaging facilitates new topical drug development process by determining quantitative skin distribution profiles

AbstractGeneration of skin distribution profiles and reliable determination of drug molecule concentration in the target region are crucial during the development process of topical products for treatment of skin diseases like psoriasis and atopic dermatitis. Imaging techniques like mass spectrometric imaging (MSI) offer sufficient spatial resolution to generate meaningful distribution profiles of a drug molecule across a skin section. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to generate quantitative skin distribution profiles based on tissue extinction coefficient (TEC) determinations of four different molecules in cross sections of human skin explants after topical administration. The four drug molecules: roflumilast, tofacitinib, ruxolitinib, and LEO 29102 have different physicochemical properties. In addition, tofacitinib was administrated in two different formulations. The study reveals that with MALDI-MSI, we were able to observe differences in penetration profiles for both the four drug molecules and the two formulations and thereby demonstrate its applicability as a screening tool when developing a topical drug product. Furthermore, the study reveals that the sensitivity of the MALDI-MSI techniques appears to be inversely correlated to the drug molecules’ ability to bind to the surrounding tissues, which can be estimated by their Log D values. Graphical abstract

Automated Morphological and Morphometric Analysis of Mass Spectrometry Imaging Data: Application to Biomarker Discovery

AbstractMass spectrometry imaging datasets are mostly analyzed in terms of average intensity in regions of interest. However, biological tissues have different morphologies with several sizes, shapes, and structures. The important biological information, contained in this highly heterogeneous cellular organization, could be hidden by analyzing the average intensities. Finding an analytical process of morphology would help to find such information, describe tissue model, and support identification of biomarkers. This study describes an informatics approach for the extraction and identification of mass spectrometry image features and its application to sample analysis and modeling. For the proof of concept, two different tissue types (healthy kidney and CT-26 xenograft tumor tissues) were imaged and analyzed. A mouse kidney model and tumor model were generated using morphometric – number of objects and total surface – information. The morphometric information was used to identify m/z that have a heterogeneous distribution. It seems to be a worthwhile pursuit as clonal heterogeneity in a tumor is of clinical relevance. This study provides a new approach to find biomarker or support tissue classification with more information. Graphical Abstractᅟ

Abstract 2064: Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft

Introduction Mass Spectrometry Imaging (MSI) is an emerging technique in preclinical analysis that allows for analysis of the distribution and the quantification of target molecules in tissue sections. The main advantage of this imaging technology is the detection of the molecule of interest in tissues without labelling and with high specificity. MSI is now routinely used for small molecule distribution analysis in lead optimization process, and in PK/PD or toxicity studies. Due to the mass range limitation of the instrument analyzer one of the main challenges is the detection of administered large molecules such as therapeutic antibodies. . In this study, we describe the detection and the distribution of trastuzumab, a monoclonal antibody that recognizes the HER2/neu receptor, in a BT474 human breast xenograft model. Experimental procedures BT474 tumor fragments are implanted in female CB.17 SCID mice. When the tumors reach 80-120 mm3, the trastuzumab treatment of 20mg/kg with eight bi-weekly intraperitoneal injections was initiated. The endpoint was fixed at day 30 post first administration. At 50 hours post 1st, 5th and 8th doses vehicle and trastuzumab treated animals were sacrificed and tumor, plasma and carcass were collected. One section of each tumor tissue was first digested with a proteolytic enzyme deposited with a dedicated device onto the tissue. After the proteolytic digestion, the selected MALDI matrix is sprayed onto the tissue and then the distribution of the Trastuzumab is determined based on its specific peptide mass fingerprint (PMF) by high resolution mass spectrometry imaging with a MALDI-FTICR instrument (SolariX FTICR 7.0T from Bruker). The images are obtained with Quantinetix software dedicated to the MSI data set. Novel Aspect We describe a process which combines MSI and bottom-up proteomics approach directly on tissue to follow the distribution of a therapeutic antibody without any labelling. Until now, mass spectrometry imaging was used to study some biomarkers9 (proteins) distribution on tissue. This work describes a novel MSI application for analysis of a dosed therapeutic antibody. Data collection is ongoing. Conclusions We have adapted the use of Mass Spectrometry Imaging to follow the distribution of a non-labeled therapeutic antibody based on its peptide mass fingerprint in dosed tissues. This opens the use of this technology to other large molecules, such as therapeutic proteins or Antibody-Drug Conjugates (ADCs) in a variety of therapeutic fields (Oncology, Immunology, CNS, etc.) In addition to the distribution of the target therapeutic antibody, Mass Spectrometry Imaging could also allow the detection of other important endogenous molecules in the same or adjacent tissue section, such as specific disease markers, thus allowing further understanding of PK/PD relationships. Citation Format: David Bonnel, Chassidy Hall, Robert J. Mullin, Kathryn A. Simon, Jonathan Stauber. Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2014-2064