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Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE-TMAs using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE-TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis.

An approach to fuse images from imaging mass spectrometry and microscopy provides biological insights into molecular tissue distributions beyond what can be obtained from either modality individually. We describe a predictive imaging modality created by 'fusing' two distinct technologies: imaging mass spectrometry (IMS) and microscopy. IMS-generated molecular maps, rich in chemical information but having coarse spatial resolution, are combined with optical microscopy maps, which have relatively low chemical specificity but high spatial information. The resulting images combine the advantages of both technologies, enabling prediction of a molecular distribution both at high spatial resolution and with high chemical specificity. Multivariate regression is used to model variables in one technology, using variables from the other technology. We demonstrate the potential of image fusion through several applications: (i) 'sharpening' of IMS images, which uses microscopy measurements to predict ion distributions at a spatial resolution that exceeds that of measured ion images by ten times or more; (ii) prediction of ion distributions in tissue areas that were not measured by IMS; and (iii) enrichment of biological signals and attenuation of instrumental artifacts, revealing insights not easily extracted from either microscopy or IMS individually.

Combining image analysis with mass spectrometry techniques provides new possibilities for molecular pathology. What are the applications of imaging mass spectrometry (IMS) using matrix-assisted laser desorption ionization (MALDI)? 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.

Imaging MS (IMS) is an emerging technology that permits the direct analysis and determination of the distribution of molecules in tissue sections. Biological molecules such as proteins, peptides, lipids, xenobiotics, and metabolites can be analyzed in a high-throughput manner with molecular specificity not readily achievable through other means. Tissues are analyzed intact and thus spatial localization of molecules within a tissue is preserved. Several studies are presented that focus on the unique types of information obtainable by IMS, such as Aβ isoform distributions in Alzheimer's plaques, protein maps in mouse brain, and spatial protein distributions in human breast carcinoma. The analysis of a biopsy taken 100 years ago from a patient with amyloidosis illustrates the use of IMS with formalin-fixed tissues. Finally, the registration and correlation of IMS with MRI is presented.

The molecular complexity of biological tissue and the spatial and temporal variation in the biological processes involved in human disease requires new technologies and new approaches to provide insight into disease processes. Imaging mass spectrometry is an effective tool that provides molecular images of tissues in the molecular discovery process. The analysis of human tissue presents special challenges and limitations because the heterogeneity among human tissues and diseases is much greater than that observed in animal models, and discoveries made in animal tissues might not translate well to their human counterparts. In this article, we briefly review the challenges of imaging human tissue using mass spectrometry and suggest approaches to address these issues.

Dietary zinc supplements are in common use, but their effect on infection is unclear. New findings now show that excess dietary zinc reduces the diversity of the gut microbiota and increases the susceptibility of antibiotic-treated mice to Clostridium difficile infection. Clostridium difficile is the most commonly reported nosocomial pathogen in the United States and is an urgent public health concern worldwide 1 . Over the past decade, incidence, severity and costs associated with C. difficile infection (CDI) have increased dramatically 2 . CDI is most commonly initiated by antibiotic-mediated disruption of the gut microbiota; however, non-antibiotic-associated CDI cases are well documented and on the rise 3 , 4 . This suggests that unexplored environmental, nutrient and host factors probably influence CDI. Here we show that excess dietary zinc (Zn) substantially alters the gut microbiota and, in turn, reduces the minimum amount of antibiotics needed to confer susceptibility to CDI. In mice colonized with C. difficile , excess dietary Zn severely exacerbated C. difficile– associated disease by increasing toxin activity and altering the host immune response. In addition, we show that the Zn-binding S100 protein calprotectin has antimicrobial effects against C. difficile and is an essential component of the innate immune response to CDI. Taken together, these data suggest that nutrient Zn levels have a key role in determining susceptibility to CDI and severity of disease, and that calprotectin-mediated metal limitation is an important factor in the host immune response to C. difficile .

Imaging mass spectrometry (IMS) has become a valuable tool for the production of molecular maps in samples ranging from solid inorganic materials to biologicals such as cells and tissues. The unique features of IMS are its ability to map a wide variety of different types of molecules, its superb molecular specificity, and its potential for discovery since no target-specific reagents are needed. IMS has made significant contributions in biology and medicine and promises to be a next generation tool in anatomic pathology. Graphical Abstract ᅟ

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R 2 ) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%. Graphical Abstract ᅟ

A prototype matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer has been used for high-speed ion image acquisition. The instrument incorporates a Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz and continuous laser raster sampling for high-throughput data collection. Lipid ion images of a sagittal rat brain tissue section were collected in 10 min with an effective acquisition rate of roughly 30 pixels/s. These results represent more than a 10-fold increase in throughput compared with current commercially available instrumentation. Experiments aimed at improving conditions for continuous laser raster sampling for imaging are reported, highlighting proper laser repetition rates and stage velocities to avoid signal degradation from significant oversampling. As new high spatial resolution and large sample area applications present themselves, the development of high-speed microprobe MALDI imaging mass spectrometry is essential to meet the needs of those seeking new technologies for rapid molecular imaging.

Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.