Jeremy L. Norris

Analysis of Tissue Specimens by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry in Biological and Clinical Research

Human beings are adept at discerning relevant information from complex systems by processing visual information. Similarly, as scientists labor to understand the fundamental nature of complex biological systems, they have continued to rely on visual information in the form of images to characterize and classify natural phenomena. New technologies designed to produce images of biological specimens have played a key role in the development of our modern understanding of biology. One of the earliest technological examples, the application of light microscopy to the analysis of biological tissue in the 17th century, ultimately led to the discovery of the cell as a key component of biology.1 Fortunately, the ways in which scientists now visualize biological systems have significantly matured. Currently, the methods for imaging biological specimens encompass an extraordinarily large range of technologies, capitalizing on many different measurable physical phenomena to produce images that provide insight into the underlying biology within the specimen. During the previous century, many imaging technologies including microscopy, radiography, ultrasonography, and magnetic resonance imaging have contributed greatly to the visualization of biological processes and to the practice of medicine.2 Each imaging modality has unique advantages and disadvantages that enable them to make contributions to research and clinical practice. One key aspect of imaging that remains a challenge is the effective integration of molecularly specific information as part of the image. Many of the commonly used in vivo imaging technologies produce high quality images, but these cannot be expressed as individual molecular images. Although immunostaining can be used to localize specific molecules within a biological sample, this method depends upon the use of a surrogate marker of the molecule such as an antibody or other specialized reagent and is usually performed on one or at most only a few molecules of interest in a single experiment. Mass spectrometry (MS) is unique among analytical technologies in its ability to directly measure individual molecular species in complex samples, allowing it to make significant contributions to our understanding of biological molecules. Indeed, the fundamental basis of the dynamic state of living systems was discovered by Rittenberg and Schoenheimer in the 1930s and 1940s through the use of MS and stable isotope tracers.3–5 With the introduction of ionization techniques such as electrospray ionization (ESI)6 and matrix-assisted laser desorption/ionization (MALDI),7 the field of mass spectrometry has grown exponentially in the past 20 years due to the application of MS to biological molecules. These capabilities ushered in a new era of biological research wherein a systems approach can be used to analyze the molecules in living systems in the wake of information provided by the Human Genome Project.8 With the drive to discover new biology has come a concomitant drive for the development of new mass spectrometry instrumentation. The primary benefit of this technology innovation is the ability to measure specific molecular compounds at high structural fidelity with high speed of acquisition, making it possible to perform experiments on biological systems that have not been possible before. Even single experiments have shown near comprehensive coverage of entire proteomes of simple organisms.9–10 Imaging Mass Spectrometry (IMS) is a technology that makes regiospecific molecular measurements directly from biological specimens.11–15 This method of imaging capitalizes on all the advantages of modern mass spectrometers, including high sensitivity, high throughput, and molecular specificity, to produce images that visually represent tissue biology on the basis of specific molecules (e.g. peptides, proteins, lipids, drugs and metabolites). The capabilities of mass spectrometry are unique in the imaging world, providing unique insights into biological systems. The distinguishing principle of imaging mass spectrometry from other mass spectrometric techniques is that the preparation of the sample and the acquisition of the MS data must be performed in a manner that preserves the spatial integrity of the sample within the limits of the spatial resolution of the measurement. Therefore, IMS of a biological sample, such as a tissue section, requires that the mass spectral data be registered to specific spatial locations in order to correlate the molecular information to specific cells or groups of cells commonly visualized by microscopy. Images are reconstructed by plotting the intensities of a given ion on a coordinate system that represents the relative position of the mass spectral acquisition from the biological sample. The resulting images create a visual representation of the sample based on the specific molecular information measured from the sample itself. IMS has a number of advantages relative to other imaging techniques currently used for biological and clinical studies. First, MS can be used to detect analytes without the need for labeling or otherwise structurally modifying the native compound. This distinction is important for many reasons, but primarily this avoids potential problems if the tagging reagent affects or changes the physical, chemical, or biological function of the molecules of interest or if the reagent has multiple molecular affinities. Second, MS has the capability of monitoring thousands of molecules in a single experiment. From a systems biology perspective, the advantage of the concurrent measurement of whole pathways or components in multiple pathways is crucial to understanding the function of intact cells. Among the several mass spectrometry ionization techniques that can be used to directly analyze tissues, MALDI has led the way in the development of biological and clinical applications for IMS.16–17 This report describes the essential considerations for performing MALDI IMS experiments on tissue, reviews some of the recent applications to the analysis of clinical specimens, highlights specific contributions of MALDI IMS to our understanding of biology and medicine, and discusses specific advantages and limitations of the technology. This review is not intended to be comprehensive with respect to all aspects of imaging mass spectrometry; rather it focuses on the themes that are essential to the analysis of biological and clinical tissue samples using MALDI IMS. There are excellent reviews that extensively cover both the ionization techniques used in IMS as well as the various mass analyzers that have been adapted for use in IMS and the reader is referred to these for further information.12,18–21

MALDI imaging mass spectrometry: Spatial molecular analysis to enable a new age of discovery ☆

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) combines the sensitivity and selectivity of mass spectrometry with spatial analysis to provide a new dimension for histological analyses to provide unbiased visualization of the arrangement of biomolecules in tissue. As such, MALDI IMS has the capability to become a powerful new molecular technology for the biological and clinical sciences. In this review, we briefly describe several applications of MALDI IMS covering a range of molecular weights, from drugs to proteins. Current limitations and challenges are discussed along with recent developments to address these issues. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez.

Biomarker Discovery by Imaging Mass Spectrometry Transthyretin is a Biomarker for Gentamicin-induced Nephrotoxicity in Rat

Adverse drug effects are often associated with pathological changes in tissue. An accurate depiction of the undesired affected area, possibly supported by mechanistic data, is important to classify the effects with regard to relevance for human patients. MALDI imaging MS represents a new analytical tool to directly provide the spatial distribution and the relative abundance of proteins in tissue. Here we evaluate this technique to investigate potential toxicity biomarkers in kidneys of rats that were administered gentamicin, a well known nephrotoxicant. Differential analysis of the mass spectrum profiles revealed a spectral feature at 12,959 Da that strongly correlates with histopathology alterations of the kidney. We unambiguously identified this spectral feature as transthyretin (Ser28–Gln146) using an innovative combination of tissue microextraction and fractionation by reverse-phase liquid chromatography followed by a top-down tandem mass spectrometric approach. Our findings clearly demonstrate the emerging role of imaging MS in the discovery of toxicity biomarkers and in obtaining mechanistic insights concerning toxicity mechanisms.

Direct imaging of single cells and tissue at sub-cellular spatial resolution using transmission geometry MALDI MS

Discussions about MALDI imaging frequently turn to the topic of spatial resolution and the eff orts of some researchers in the field to push towards routine imaging of tissue sections at a cellular scale. Some factors that limit resolution are, the size of the focused desorption laser beam and analyte delocalization from the solution-based sample preparation. With solvent-free matrix application techniques analyte delocalization is less of a concern and the size of the focused laser is the major limiter of spatial resolution. In the Special Feature, Professor Caprioli and co-workers at Vanderbilt University demonstrate a new instrumental approach for improving spatial resolution. They have modifi ed a MALDI-TOF system to use transmission-mode geometry, in which the desorption laser is focused onto the matrix crystals from behind and through the target and sample rather than conventional front-side illumination where the laser is focused onto the crystals directly. They show that by moving the laser source behind the sample target, they can optimize the laser focus to achieve cellular resolution for MALDI imaging.

Direct imaging of single cells and tissue at sub-cellular spatial resolution using transmission geometry MALDI MS: Single-cell imaging with transmission geometry

The need of cellular and sub-cellular spatial resolution in laser desorption ionization (LDI)/matrix-assisted LDI (MALDI) imaging mass spectrometry (IMS) necessitates micron and sub-micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end, we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical/ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub-cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub-micron spatial resolution.

Phase II Study of Neoadjuvant Imatinib in Glioblastoma: Evaluation of Clinical and Molecular Effects of the Treatment

Purpose: Phase I-II studies indicate that imatinib is active in glioblastoma multiforme. To better understand the molecular and clinical effects of imatinib in glioblastoma multiforme, we conducted a neoadjuvant study of imatinib with pretreatment and posttreatment biopsies. Experimental Design: Patients underwent a computerized tomography-guided biopsy of their brain tumors. If diagnosed with glioblastoma multiforme, they were immediately treated with 7 days of imatinib 400 mg orally twice daily followed by either definitive surgery or re-biopsy. Pretreatment and posttreatment tissue specimens were tested by immunohistochemistry for Ki67 and microvessel destiny, and posttreatment specimens were analyzed for the presence of intact imatinib in tissue. Furthermore, pretreatment and posttreatment pairs were analyzed by Western blotting for activation of platelet-derived growth factor receptor, epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase signaling pathways. Pharmacokinetic studies were also done. Results: Twenty patients were enrolled. Median survival was 6.2 months. Intact imatinib was detected in the posttreatment tissue specimens using mass spectrometry. There was no evidence of a drug effect on proliferation, as evidenced by a change in Ki67 expression. Biochemical evidence of response, as shown by decreased activation of AKT and mitogen-activated protein kinase or increased p27 level, was detected in 4 of 11 patients with evaluable, matched pre- and post-imatinib biopsies. Two patients showed high-level EGFR activation and homozygous EGFR mutations, whereas one patient had high-level platelet-derived growth factor receptor-B activation. Conclusions: Intact imatinib was detected in glioblastoma multiforme tissue. However, the histologic and immunoblotting evaluations suggest that glioblastoma multiforme proliferation and survival mechanisms are not substantially reduced by imatinib therapy in most patients. (Clin Cancer Res 2009;15(19):6258–66)

Robust Analysis of the Yeast Proteome under 50 kDa by Molecular-Mass-Based Fractionation and Top-Down Mass Spectrometry

As the process of top-down mass spectrometry continues to mature, we benchmark the next installment of an improving methodology that incorporates a tube-gel electrophoresis (TGE) device to separate intact proteins by molecular mass. Top-down proteomics is accomplished in a robust fashion to yield the identification of hundreds of unique proteins, many of which correspond to multiple protein forms. The TGE platform separates 0-50 kDa proteins extracted from the yeast proteome into 12 fractions prior to automated nanocapillary LC-MS/MS in technical triplicate. The process may be completed in less than 72 h. From this study, 530 unique proteins and 1103 distinct protein species were identified and characterized, thus representing the highest coverage to date of the Saccharomyces cerevisiae proteome using top-down proteomics. The work signifies a significant step in the maturation of proteomics based on direct measurement and fragmentation of intact proteins.

Imaging mass spectrometry: a new tool for pathology in a molecular age.

Mass spectrometry (MS) provides unique advantages for the analysis of clinical specimens, and these capabilities have been critical to the advancement of diagnostic medicine. To date, LC‐MS is the MS platform most commonly used for diagnostics; however, LC‐MS based proteomics is very labor intensive and costly to implement for high volume assays. Furthermore, when analyzing tissue samples, additional laborious sample preparation steps must be employed (e.g. extraction methods or laser microdissection). The direct analysis of cells and tissues by MALDI imaging MS has developed significant momentum for applications that have diagnostic potential. MALDI imaging MS provides molecular information from specific cell types within tissue sections; however, this laser‐based approach significantly reduces the analysis time for each location sampled. This Viewpoint discusses the technologies for direct analysis of tissues, the potential for diagnostic applications using MALDI imaging MS, and the challenges faced in the transfer of the technology to the clinical laboratory.

A Derivatization and Validation Strategy for Determining the Spatial Localization of Endogenous Amine Metabolites in Tissues using MALDI Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MS(n) IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds.

Direct sampling and analysis from solid-phase extraction cards using an automated liquid extraction surface analysis nanoelectrospray mass spectrometry system †

Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as a liquid extraction surface analysis (LESA) mode on a commercially available chip-based infusion nanoelectrospray ionization (nanoESI) system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom-made solid-phase extraction (SPE) cards, with each well consisting of either a 1 or a 2 mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the commercial nanoESI system coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer or a linear ion trap mass spectrometer. The extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10× increase in signal and cleanup of micromolar angiotensin II from a concentrated salt solution was demonstrated. In addition, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS(3) spectra for compound identification in the presence of isobaric interferences.