Thermal Denaturation of Fresh Frozen Tissue Enhances Mass Spectrometry Detection of Peptides.

Thermal denaturation (TD), known as antigen retrieval, heats tissue samples in a buffered solution to expose protein epitopes. Thermal denaturation of formalin-fixed paraffin-embedded samples enhances on-tissue tryptic digestion, increasing peptide detection using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS). We investigated the tissue-dependent effects of TD on peptide MALDI IMS and liquid chromatography-tandem mass spectrometry signal in unfixed, frozen human colon, ovary, and pancreas tissue.

Processes in DNA damage response from a whole-cell multi-omics perspective.

Technological advances have made it feasible to collect multi-condition multi-omic time courses of cellular response to perturbation, but the complexity of these datasets impedes discovery due to challenges in data management, analysis, visualization, and interpretation. Here, we report a whole-cell mechanistic analysis of HL-60 cellular response to bendamustine. We integrate both enrichment and network analysis to show the progression of DNA damage and programmed cell death over time in molecular, pathway, and process-level detail using an interactive analysis framework for multi-omics data.

Rapid Multivariate Analysis Approach to Explore Differential Spatial Protein Profiles in Tissue.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from -infected murine kidney, 4 and 10 days postinfection.

Enhancement of Tryptic Peptide Signals from Tissue Sections Using MALDI IMS Postionization (MALDI-2).

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve the sensitivity and depth of coverage for protein and peptide IMS. We demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (MALDI-1) augmented using a second ionizing laser (termed MALDI-2).

Protocol for multimodal analysis of human kidney tissue by imaging mass spectrometry and CODEX multiplexed immunofluorescence.

Here, we describe the preservation and preparation of human kidney tissue for interrogation by histopathology, imaging mass spectrometry, and multiplexed immunofluorescence. Custom image registration and integration techniques are used to create cellular and molecular atlases of this organ system. Through careful optimization, we ensure high-quality and reproducible datasets suitable for cross-patient comparisons that are essential to understanding human health and disease.

Spatially Targeted Proteomics of the Host-Pathogen Interface during Staphylococcal Abscess Formation.

is a common cause of invasive and life-threatening infections that are often multidrug resistant. To develop novel treatment approaches, a detailed understanding of the complex host-pathogen interactions during infection is essential. This is particularly true for the molecular processes that govern the formation of tissue abscesses, as these heterogeneous structures are important contributors to staphylococcal pathogenicity.

A Multiplex Analysis of Potentially Toxic Cyanobacteria in Lake Winnipeg during the 2013 Bloom Season.

Lake Winnipeg (Manitoba, Canada), the world's 12th largest lake by area, is host to yearly cyanobacterial harmful algal blooms (cHABs) dominated by and . cHABs in Lake Winnipeg are primarily a result of eutrophication but may be exacerbated by the recent introduction of dreissenid mussels. Through multiple methods to monitor the potential for toxin production in Lake Winnipeg in conjunction with environmental measures, this study defined the baseline composition of a Lake Winnipeg cHAB to measure potential changes because of dreissenid colonization.

A recommended and verified procedure for in situ tryptic digestion of formalin-fixed paraffin-embedded tissues for analysis by matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology.

Zinc intoxication induces ferroptosis in A549 human lung cells.

Zinc (Zn) is an essential trace metal required for all forms of life, but is toxic at high concentrations. While the toxic effects of high levels of Zn are well documented, the mechanism of cell death appears to vary based on the study and concentration of Zn. Zn has been proposed as an anti-cancer treatment against non-small cell lung cancer (NSCLC).

An Integrated, High-Throughput Strategy for Multiomic Systems Level Analysis.

Proteomics, metabolomics, and transcriptomics generate comprehensive data sets, and current biocomputational capabilities allow their efficient integration for systems biology analysis. Published multiomics studies cover methodological advances as well as applications to biological questions. However, few studies have focused on the development of a high-throughput, unified sample preparation approach to complement high-throughput omic analytics.

Micro-Data-Independent Acquisition for High-Throughput Proteomics and Sensitive Peptide Mass Spectrum Identification.

State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (μDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the μDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH.

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995.

Inhibitory effects of fenretinide metabolites N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR) on fenretinide molecular targets β-carotene oxygenase 1, stearoyl-CoA desaturase 1 and dihydroceramide Δ4-desaturase 1.

The therapeutic capacity of fenretinide (N-[4-hydroxyphenyl] retinamide; 4-HPR) has been demonstrated for several conditions, including cancer, obesity, diabetes, and ocular disease. Yet, the mechanisms of action for its pleiotropic effects are still undefined. We hypothesized that investigation of two of the major physiological metabolites of fenretinide, N-[4-methoxyphenyl]retinamide (MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), might begin to resolve the multifaceted effects of this synthetic retinoid.

Euglenophycin is produced in at least six species of euglenoid algae and six of seven strains of Euglena sanguinea.

Euglena sanguinea is known to produce the alkaloid toxin euglenophycin and is known to cause fish kills and inhibit mammalian tissue and microalgal culture growth. An analysis of over 30 species of euglenoids for accumulation of euglenophycin identified six additional species producing the toxin; and six of the seven E. sanguinea strains produced the toxin.

Integrated, High-Throughput, Multiomics Platform Enables Data-Driven Construction of Cellular Responses and Reveals Global Drug Mechanisms of Action.

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.

Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses.

In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens.

Pathology interface for the molecular analysis of tissue by mass spectrometry.

Background: Imaging mass spectrometry (IMS) generates molecular images directly from tissue sections to provide better diagnostic insights and expand the capabilities of clinical anatomic pathology. Although IMS technology has matured over recent years, the link between microscopy imaging currently used by pathologists and MS-based molecular imaging has not been established.

Methods: We adapted the Vanderbilt University Tissue Core workflow for IMS into a web-based system that facilitates remote collaboration.

Imaging mass spectrometry of the visual system: Advancing the molecular understanding of retina degenerations.

Visual sensation is fundamental for quality of life, and loss of vision to retinal degeneration is a debilitating condition. The eye is the only part of the central nervous system that can be noninvasively observed with optical imaging. In the clinics, various spectroscopic methods provide high spatial resolution images of the fundus and the developing degenerative lesions.

Phosphatidylcholine composition of pulmonary surfactant from terrestrial and marine diving mammals.

Marine mammals are repeatedly exposed to elevated extra-thoracic pressure and alveolar collapse during diving and readily experience alveolar expansion upon inhalation - a unique capability as compared to terrestrial mammals. How marine mammal lungs overcome the challenges of frequent alveolar collapse and recruitment remains unknown. Recent studies indicate that pinniped lung surfactant has more anti-adhesive components compared to terrestrial mammals, which would aid in alveolar opening.

Quantitative autofluorescence and cell density maps of the human retinal pigment epithelium.

Purpose: Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF.

Methods: Retinal pigment epithelium-Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.

Quantitative mass spectrometric analysis and post-extraction stability assessment of the euglenoid toxin euglenophycin.

Euglenophycin is a recently discovered toxin produced by at least one species of euglenoid algae. The toxin has been responsible for several fish mortality events. To facilitate the identification and monitoring of euglenophycin in freshwater ponds, we have developed a specific mass spectrometric method for the identification and quantitation of euglenophycin.

Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.

Purpose: The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE.

Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling.

Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs).

Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0.