The identification of proteins from tissue specimens is a challenging area of biological research. Many current techniques for identification forfeit some level of spatial information during the sample preparation process. Recently, hydrogel technologies have been developed that perform spatially localized protein extraction and digestion prior to downstream proteomic analysis. Regiospecific protein identifications acquired using this approach have thus far been limited to 1–2 mm diameter areas. The need to target smaller populations of cells with this technology necessitates the production of smaller diameter hydrogels. Herein, we demonstrate hydrogel fabrication processes that allow hydrogel applications down to a diameter of ∼260 μm, approximately 1/15 of the area of previous approaches. Parameters such as the percent polyacrylamide used in hydrogel construction as well as the concentration of trypsin with which the hydrogel is loaded are investigated to maximize the number of protein identifications from subsequent liquid chromatography tandem MS (LC–MS/MS) analysis of hydrogel extracts. An 18% polyacrylamide concentration is shown to provide for a more rigid polymer network than the conventional 7.5% polyacrylamide concentration and supports the fabrication of individual hydrogels using the small punch biopsies. Over 600 protein identifications are still achieved at the smallest hydrogel diameters of 260 μm. The utility of these small hydrogels is demonstrated through the analysis of sub regions of a rat cerebellum tissue section. While over 900 protein identifications are made from each hydrogel, approximately 20% of the proteins identified are unique to each of the two regions, highlighting the importance of targeting tissue subtypes to accurately characterize tissue biology. These newly improved methods to the hydrogel process will allow researchers to target smaller biological features for robust spatially localized proteomic analyses.

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10 000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.Keywords: cisplatin; drug discovery; high-throughput; mechanism of action; omics;

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 (R2) 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%.

Imaging mass spectrometry (IMS) is a molecular imaging technology that can measure thousands of biomolecules concurrently without prior tagging, making it particularly suitable for exploratory research. However, the data size and dimensionality often makes thorough extraction of relevant information impractical. To help guide and accelerate IMS data analysis, we recently developed a framework that integrates IMS measurements with anatomical atlases, opening up opportunities for anatomy-driven exploration of IMS data. One example is the automated anatomical interpretation of ion images, where empirically measured ion distributions are automatically decomposed into their underlying anatomical structures.While offering significant potential, IMS-atlas integration has thus far been restricted to the Allen Mouse Brain Atlas (AMBA) and mouse brain samples. Here, we expand the applicability of this framework by extending towards new animal species and a new set of anatomical atlases retrieved from the Scalable Brain Atlas (SBA). Furthermore, as many SBA atlases are based on magnetic resonance imaging (MRI) data, a new registration pipeline was developed that enables direct non-rigid IMS-to-MRI registration. These developments are demonstrated on protein-focused FTICR IMS measurements from coronal brain sections of a Parkinson's disease (PD) rat model. The measurements are integrated with an MRI-based rat brain atlas from the SBA. The new rat-focused IMS-atlas integration is used to perform automated anatomical interpretation and to find differential ions between healthy and diseased tissue. IMS-atlas integration can serve as an important accelerator in IMS data exploration, and with these new developments it can now be applied to a wider variety of animal species and modalities. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

In this review, we will highlight technologies that enable scientists to study the molecular characteristics of tissues and/or cells without the need for antibodies or other labeling techniques. Specifically, we will focus on matrix-assisted laser desorption/ionization imaging mass spectrometry, infrared spectroscopy, and Raman spectroscopy.

Quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) approaches have historically suffered from poor accuracy and precision mainly due to the nonuniform distribution of matrix and analyte across the target surface, matrix interferences, and ionization suppression. Tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity as well as improve signal-to-noise ratios by eliminating interferences from chemical noise, alleviating some concerns about dynamic range. However, conventional MALDI TOF/TOF modalities typically only scan for a single MS/MS event per laser shot, and multiplex assays require sequential analyses. We describe here new methodology that allows for multiple TOF/TOF fragmentation events to be performed in a single laser shot. This technology allows the reference of analyte intensity to that of the internal standard in each laser shot, even when the analyte and internal standard are quite disparate in m/z, thereby improving quantification while maintaining chemical specificity and duty cycle. In the quantitative analysis of the drug enalapril in pooled human plasma with ramipril as an internal standard, a greater than 4-fold improvement in relative standard deviation (<10%) was observed as well as improved coefficients of determination (R2) and accuracy (>85% quality controls). Using this approach we have also performed simultaneous quantitative analysis of three drugs (promethazine, enalapril, and verapamil) using deuterated analogues of these drugs as internal standards.

A simultaneous on-tissue proteolytic digestion and extraction method is described for the in situ analysis of proteins from spatially distinct areas of a tissue section. The digestion occurs on-tissue within a hydrogel network, and peptides extracted from this gel are identified with liquid chromatography tandem MS (LC-MS/MS). The hydrogels are compatible with solubility agents (e.g., chaotropes and detergents) known to improve enzymatic digestion of proteins. Additionally, digestions and extractions are compatible with imaging mass spectrometry (IMS) experiments. As an example application, an initial IMS experiment was conducted to profile lipid species using a traveling wave ion mobility mass spectrometer. On-tissue MS/MS was also performed on the same tissue section to identify lipid ions that showed spatial differences. Subsequently, the section underwent an on-tissue hydrogel digestion to reveal 96 proteins that colocalized to the rat brain cerebellum. Hematoxylin and eosin (H & E) staining was then performed to provide additional histological information about the tissue structure. This technology provides a versatile workflow that can be used to correlate multiple complementary analytical approaches in the analysis of a single tissue section.

We have achieved enhanced lipid imaging to a ∼10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix, and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to 5-fold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution.

A precise mixture of extracellular matrix (ECM) secreted by valvular cells forms a scaffold that lends the heart valve the exact mechanical and tensile strength needed for accurate hemodynamic performance. ECM proteins are a key component of valvular endothelial cell (VEC)−valvular interstitial cell (VIC) communication essential for maintenance of the valve structure. This study reports the healthy adult pulmonary and aortic valve proteomes characterized by LC−MS/MS, resulting in 2710 proteins expressed by 1513 genes, including over 300 abundant ECM proteins. Surprisingly, this study defines a distinct proteome for each semilunar valve. Protein−protein networking (PPN) was used as a tool to direct selection of proteomic candidates for biological investigation. Local PPN for nidogen 1 (Nid1), biglycan (Bgn), elastin microfibril interface-located protein 1 (Emilin-1), and milk fat globule-EGF factor 8 protein (Mfge8) were enriched with proteins essential to valve function and produced biological functions highly relevant to valve biology. Immunofluorescent investigations demonstrated that these proteins are functionally distributed within the pulmonary and aortic valve structure, indicative of important contribution to valve function. This study yields new insight into protein expression contributing to valvular maintenance and health and provides a platform for unbiased assessment of protein alterations during disease processes.

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.

MALDI Imaging Mass Spectrometry is a molecular analytical technology capable of simultaneously measuring multiple analytes directly from intact tissue sections. Histological features within the sample can be correlated with molecular species without the need for target-specific reagents such as antibodies. Several studies have demonstrated the strength of the technology for uncovering new markers that correlate with disease severity as well as prognosis and therapeutic response. This review describes technological aspects of imaging mass spectrometry together with applications in cancer research.