Richard M. Caprioli

Imaging mass spectrometry: A new technology for the analysis of protein expression in mammalian tissues

1and has been initially targeted for the analysis of peptides and proteins present on or near the surface of tissue sections 2 . Imaging MS brings a new tool to bear on the problem of unraveling and understanding the molecular complexities of cells. It joins techniques such as immunochemistry and fluorescence microscopy for the study of the spatial arrangement of molecules within biological tissues. Many previous experiments using MS to image samples have focused on the measurement of the distribution of elements and small molecules in biological specimens, including tissue slices and individual cells 3‐5 . An extensive review on imaging by MS can be found in the article by Pacholski and Winograd 6

MALDI imaging mass spectrometry: Molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.

Proteomic patterns of tumour subsets in non-small-cell lung cancer.

BACKGROUND Proteomics-based approaches complement the genome initiatives and may be the next step in attempts to understand the biology of cancer. We used matrix-assisted laser desorption/ionisation mass spectrometry directly from 1-mm regions of single frozen tissue sections for profiling of protein expression from surgically resected tissues to classify lung tumours. METHODS Proteomic spectra were obtained and aligned from 79 lung tumours and 14 normal lung tissues. We built a class-prediction model with the proteomic patterns in a training cohort of 42 lung tumours and eight normal lung samples, and assessed their statistical significance. We then applied this model to a blinded test cohort, including 37 lung tumours and six normal lung samples, to estimate the misclassification rate. FINDINGS We obtained more than 1600 protein peaks from histologically selected 1 mm diameter regions of single frozen sections from each tissue. Class-prediction models based on differentially expressed peaks enabled us to perfectly classify lung cancer histologies, distinguish primary tumours from metastases to the lung from other sites, and classify nodal involvement with 85% accuracy in the training cohort. This model nearly perfectly classified samples in the independent blinded test cohort. We also obtained a proteomic pattern comprised of 15 distinct mass spectrometry peaks that distinguished between patients with resected non-small-cell lung cancer who had poor prognosis (median survival 6 months, n=25) and those who had good prognosis (median survival 33 months, n=41, p<0.0001). INTERPRETATION Proteomic patterns obtained directly from small amounts of fresh frozen lung-tumour tissue could be used to accurately classify and predict histological groups as well as nodal involvement and survival in resected non-small-cell lung cancer.

Metal Chelation and Inhibition of Bacterial Growth in Tissue Abscesses

Bacterial infection often results in the formation of tissue abscesses, which represent the primary site of interaction between invading bacteria and the innate immune system. We identify the host protein calprotectin as a neutrophil-dependent factor expressed inside Staphylococcus aureus abscesses. Neutrophil-derived calprotectin inhibited S. aureus growth through chelation of nutrient Mn2+ and Zn2+: an activity that results in reprogramming of the bacterial transcriptome. The abscesses of mice lacking calprotectin were enriched in metal, and staphylococcal proliferation was enhanced in these metal-rich abscesses. These results demonstrate that calprotectin is a critical factor in the innate immune response to infection and define metal chelation as a strategy for inhibiting microbial growth inside abscessed tissue.

Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins

A “micro-electrospray” ionization source has been developed that markedly increases the sensitivity of the conventional electrospray source. This was achieved by optimization of the source to accommodate nanoliter flow rates from 300 to 800-nL/min spraying directly from a capillary needle that, for the analysis of peptides, contained C18 liquid chromatography packing as an integrated concentration-desalting device. Thus, a total of 1 fmol of methionine enkephalin was desorbed from the capillary column spray needle, loaded as a 10-μL injection of 100-amol/μL solution. The mass spectrum showed the [M + H]+ ion at m/z 574.2 with a signal-to-noise ratio of better than 5:1 from a chromatographic peak with a width of about 12 s. A narrow range (15-u) tandem mass spectrum was obtained for methionine enkephalin from the injection of 500 amol, and a full-scan tandem-mass spectrum was obtained from 50 fmol. For proteins, the average mass measurement accuracy was approximately 100–200 ppm for the injection of 2.5 fmol of apomyoglobin and 20–40 ppm for 200 fmol. Carbonic anhydrase B and bovine serum albumin showed similar mass measurement accuracies.

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-FTICR Imaging Mass Spectrometry of Drugs and Metabolites in Tissue

A new approach is described for imaging mass spectrometry analysis of drugs and metabolites in tissue using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR). The technique utilizes the high resolving power to produce images from thousands of ions measured during a single mass spectrometry (MS)-mode experiment. Accurate mass measurement provides molecular specificity for the ion images on the basis of elemental composition. Final structural confirmation of the targeted compound is made from accurate mass fragment ions generated in an external quadrupole-collision cell. The ability to image many small molecules in a single measurement with high specificity is a significant improvement over existing MS/MS based technologies. Example images are shown for olanzapine in kidney and liver and imatinib in glioma.

High-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue microarrays using MALDI imaging mass spectrometry

A novel method for high‐throughput proteomic analysis of formalin‐fixed paraffin‐embedded (FFPE) tissue microarrays (TMA) is described using on‐tissue tryptic digestion followed by MALDI imaging MS. A TMA section containing 112 needle core biopsies from lung‐tumor patients was analyzed using MS and the data were correlated to a serial hematoxylin and eosin (H&E)‐stained section having various histological regions marked, including cancer, non‐cancer, and normal ones. By correlating each mass spectrum to a defined histological region, statistical classification models were generated that can sufficiently distinguish biopsies from adenocarcinoma from squamous cell carcinoma biopsies. These classification models were built using a training set of biopsies in the TMA and were then validated on the remaining biopsies. Peptide markers of interest were identified directly from the TMA section using MALDI MS/MS sequence analysis. The ability to detect and characterize tumor marker proteins for a large cohort of FFPE samples in a high‐throughput approach will be of significant benefit not only to investigators studying tumor biology, but also to clinicians for diagnostic and prognostic purposes.

Molecular imaging by mass spectrometry — looking beyond classical histology

Imaging mass spectrometry (IMS) using matrix-assisted laser desorption ionization (MALDI) is a new and effective tool for molecular studies of complex biological samples such as tissue sections. As histological features remain intact throughout the analysis of a section, distribution maps of multiple analytes can be correlated with histological and clinical features. Spatial molecular arrangements can be assessed without the need for target-specific reagents, allowing the discovery of diagnostic and prognostic markers of different cancer types and enabling the determination of effective therapies.

Proteomics in Diagnostic Pathology: Profiling and Imaging Proteins Directly in Tissue Sections

Direct tissue profiling and imaging mass spectrometry (MS) provide a molecular assessment of numerous expressed proteins within a tissue sample. MALDI MS (matrix-assisted laser desorption ionization) analysis of thin tissue sections results in the visualization of 500 to 1000 individual protein signals in the molecular weight range from 2000 to over 200,000. These signals directly correlate with protein distribution within a specific region of the tissue sample. The systematic investigation of the section allows the construction of ion density maps, or specific molecular images, for virtually every signal detected in the analysis. Ultimately, hundreds of images, each at a specific molecular weight, may be obtained. To date, profiling and imaging MS has been applied to multiple diseased tissues, including human non-small cell lung tumors, gliomas, and breast tumors. Interrogation of the resulting complex MS data sets using modern biocomputational tools has resulted in identification of both disease-state and patient-prognosis specific protein patterns. These studies suggest that such proteomic information will become more and more important in assessing disease progression, prognosis, and drug efficacy. Molecular histology has been known for some time and its value clear in the field of pathology. Imaging mass spectrometry brings a new dimension of molecular data, one focusing on the disease phenotype. The present article reviews the state of the art of the technology and its complementarity with traditional histopathological analyses.