RESUMEN
Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.
Asunto(s)
Técnicas Biosensibles , Pruebas de Enzimas/métodos , Ingeniería Metabólica/métodos , Asteraceae/enzimología , Asteraceae/genética , Biocatálisis , Técnicas Analíticas Microfluídicas , Mutagénesis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMEN
Membrane lipids serve as substrates for the generation of numerous signaling lipids when plants are exposed to environmental stresses, and jasmonic acid, an oxidized product of 18-carbon unsaturated fatty acids (e.g., linolenic acid), has been recognized as the essential signal in wound-induced gene expression. Yet, the contribution of individual membrane lipids in linolenic acid generation is ill-defined. In this work, we performed spatial lipidomic experiments to track lipid changes that occur locally at the sight of leaf injury to better understand the potential origin of linolenic and linoleic acids from individual membrane lipids. The central veins of tomato leaflets were crushed using surgical forceps, leaves were cryosectioned and analyzed by two orthogonal matrix-assisted laser desorption/ionization mass spectrometry imaging platforms for insight into lipid spatial distribution. Significant changes in lipid composition are only observed 30 min after wounding, while after 60 min lipidome homeostasis has been re-established. Phosphatidylcholines exhibit a variable pattern of spatial behavior in individual plants. Among lysolipids, lysophosphatidylcholines strongly co-localize with the injured zone of wounded leaflets, while, for example, lysophosphatidylglycerol (LPG) (16:1) accumulated preferentially toward the apex in the injured zone of wounded leaflets. In contrast, two other LPGs (LPG [18:3] and LPG [18:2]) are depleted in the injured zone. Our high-resolution co-localization imaging analyses suggest that linolenic acids are predominantly released from PCs with 16_18 fatty acid composition along the entire leaf, while it seems that in the apex zone PG (16:1_18:3) significantly contributes to the linolenic acid pool. These results also indicate distinct localization and/or substrate preferences of phospholipase isoforms in leaf tissue.
RESUMEN
Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is an emerging analytical technique, which generates spatially resolved proteomic and metabolomic images from tissue specimens. Conventional MALDI MSI processing and data acquisition can take over 30 min, limiting its clinical utility for intraoperative diagnostics. We present a rapid MALDI MSI method, completed under 5 min, including sample preparation and analysis, providing a workflow compatible with the clinical frozen section procedure.
RESUMEN
In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production.
Asunto(s)
Proteínas Bacterianas/genética , Elementos Transponibles de ADN , Mutagénesis Insercional , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Fenazinas/metabolismo , Pseudomonas aeruginosa/química , Espectrometría de Masas en TándemRESUMEN
In polymicrobial infections, microbes can interact with both the host immune system and one another through direct contact or the secretion of metabolites, affecting disease progression and treatment options. The thick mucus in the lungs of patients with cystic fibrosis is highly susceptible to polymicrobial infections by opportunistic pathogens, including the bacterium Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. Unravelling the hidden molecular interactions within such polymicrobial communities and their metabolic exchange processes will require effective enabling technologies applied to model systems. In the present study, MALDI-TOF and MALDI-FT-ICR imaging mass spectrometry (MALDI-IMS) combined with MS/MS networking were used to provide insight into the interkingdom interaction between P. aeruginosa and A. fumigatus at the molecular level. The combination of these technologies enabled the visualization and identification of metabolites secreted by these microorganisms grown on agar. A complex molecular interplay was revealed involving suppression, increased production, and biotransformation of a range of metabolites. Of particular interest is the observation that P. aeruginosa phenazine metabolites were converted by A. fumigatus into other chemical entities with alternative properties, including enhanced toxicities and the ability to induce fungal siderophores. This work highlights the capabilities of MALDI-IMS and MS/MS network analysis to study interkingdom interactions and provides insight into the complex nature of polymicrobial metabolic exchange and biotransformations.
Asunto(s)
Espectrometría de Masas/métodos , Pseudomonas aeruginosa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Aspergillus fumigatus/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Coinfección/microbiología , Fibrosis Quística/microbiología , Humanos , Iones , Técnicas Microbiológicas , Modelos Biológicos , Pseudomonas aeruginosa/genética , Especificidad de la Especie , Espectrometría de Masas en Tándem/métodos , Factores de TiempoRESUMEN
Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or ß-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard "toolbox" of MALDI imaging for reliable results under conditions of automation.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Encéfalo/ultraestructura , Química Encefálica , Insulina/análisis , Masculino , Ratones , Páncreas/química , Páncreas/ultraestructura , Proteómica/métodos , Ratas , Testículo/química , Testículo/ultraestructuraRESUMEN
Significant progress in instrumentation and sample preparation approaches have recently expanded the potential of MALDI imaging mass spectrometry to the analysis of phospholipids and other endogenous metabolites naturally occurring in tissue specimens. Here we explore some of the requirements necessary for the successful analysis and imaging of phospholipids from thin tissue sections of various dimensions by MALDI time-of-flight mass spectrometry. We address methodology issues relative to the imaging of whole-body sections such as those cut from model laboratory animals, sections of intermediate dimensions typically prepared from individual organs, as well as the requirements for imaging areas of interests from these sections at a cellular scale spatial resolution. We also review existing limitations of MALDI imaging MS technology relative to compound identification. Finally, we conclude with a perspective on important issues relative to data exploitation and management that need to be solved to maximize biological understanding of the tissue specimen investigated.
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Fosfolípidos/química , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Encéfalo/metabolismo , Encéfalo/patología , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos ICR , ProteomaRESUMEN
Analysis of formalin-fixed paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMAs) are available, but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated matrix-assisted laser desorption ionzation mass spectrometry (MALDI MS) analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney.
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Electroforesis/métodos , Adhesión en Parafina , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis de Matrices Tisulares/métodos , Fijación del Tejido , Animales , Antígenos/análisis , Electroforesis/economía , Formaldehído , Humanos , Riñón/química , Ratones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/economíaRESUMEN
Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4-8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A(2alpha) and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.
Asunto(s)
Implantación del Embrión , Espectrometría de Masas/métodos , Fosfolípidos/análisis , Animales , Ciclooxigenasa 2/metabolismo , Citosol/enzimología , Femenino , Fosfolipasas A2 Grupo IV/metabolismo , Inmunohistoquímica , Masculino , Ratones , Fosfatidilcolinas/análisis , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análisis , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositoles/análisis , Fosfatidilinositoles/metabolismo , Fosfolípidos/metabolismo , Embarazo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingomielinas/análisis , Esfingomielinas/metabolismo , Factores de Tiempo , Útero/metabolismoRESUMEN
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.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Hígado/metabolismo , Masculino , Ratones , Estructura Molecular , Preparaciones Farmacéuticas/química , RatasRESUMEN
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.
Asunto(s)
Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Biología Evolutiva/métodos , Patología/métodos , Preparaciones Farmacéuticas/metabolismo , Fosfolípidos/metabolismo , Proteómica/tendencias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/tendenciasRESUMEN
Profiling and imaging biological specimens using MALDI mass spectrometry has significant potential to contribute to our understanding and diagnosis of disease. The technique is efficient and high-throughput providing a wealth of data about the biological state of the sample from a very simple and direct experiment. However, in order for these techniques to be put to use for clinical purposes, the approaches used to process and analyze the data must improve. This study examines some of the existing tools to baseline subtract, normalize, align, and remove spectral noise for MALDI data, comparing the advantages of each. A preferred workflow is presented that can be easily implemented for data in ASCII format. The advantages of using such an approach are discussed for both molecular profiling and imaging mass spectrometry.
RESUMEN
We describe a novel tissue profiling strategy that improves the cellular specificity and analysis throughput of protein profiles obtained by direct MALDI analysis. The new approach integrates the cellular specificity of histology, the accuracy and reproducibility of robotic liquid dispensing, and the speed and objectivity of automated spectra acquisition. Traditional methodologies for preparing and analyzing tissue samples rely heavily on manual procedures, which for various reasons discussed, restrict cellular specificity and sample throughput. Here, a robotic spotter deposits micron-sized droplets of matrix precisely onto foci of normal mammary epithelium, ductal carcinoma in situ, invasive mammary cancer, and peritumoral stroma selected by a pathologist from high resolution histological images of sectioned human breast cancer samples. The location of each matrix spot was then determined and uploaded into the instrument to facilitate automated profile acquisition by MALDI-TOF. In the example shown, the different lesions were clearly differentiated using mass profiling. Further, the workflow permits a visual projection of any information produced from the profile analyses directly on the histological image for a unique combination of proteomic and histological assessment of sample regions. The higher performance characteristics offered by the new workflow promises to be a significant advancement toward the next generation of tissue profiling studies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis de Matrices Tisulares , Adulto , Animales , Femenino , Humanos , Ratones , Ratones Transgénicos , Microdisección , Persona de Mediana Edad , Proteínas de Neoplasias/químicaRESUMEN
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 (Ser(28)-Gln(146)) 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.
Asunto(s)
Gentamicinas/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Riñón/efectos de los fármacos , Prealbúmina/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Biomarcadores/análisis , Biomarcadores/química , Western Blotting , Riñón/citología , Riñón/patología , Enfermedades Renales/patología , Masculino , Datos de Secuencia Molecular , Prealbúmina/química , Análisis por Matrices de Proteínas , Ratas , Ratas Wistar , Reproducibilidad de los ResultadosRESUMEN
Novel high-throughput sample preparation strategies for MALDI imaging mass spectrometry (IMS) and profiling are presented. An acoustic reagent multispotter was developed to provide improved reproducibility for depositing matrix onto a sample surface, for example, such as a tissue section. The unique design of the acoustic droplet ejector and its optimization for depositing matrix solution are discussed. Since it does not contain a capillary or nozzle for fluid ejection, issues with clogging of these orifices are avoided. Automated matrix deposition provides better control of conditions affecting protein extraction and matrix crystallization with the ability to deposit matrix accurately onto small surface features. For tissue sections, matrix spots of 180-200 microm in diameter were obtained and a procedure is described for generating coordinate files readable by a mass spectrometer to permit automated profile acquisition. Mass spectral quality and reproducibility was found to be better than that obtained with manual pipet spotting. The instrument can also deposit matrix spots in a dense array pattern so that, after analysis in a mass spectrometer, two-dimensional ion images may be constructed. Example ion images from a mouse brain are presented.