RESUMEN
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. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.
Asunto(s)
Proteínas/análisis , Manejo de Especímenes/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Análisis de Matrices Tisulares/métodos , Formaldehído/química , Humanos , Adhesión en Parafina , Proteolisis , Fijación del Tejido , Tripsina/químicaRESUMEN
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). The goal of this analysis was to determine the effects of Zn on metabolism and cell death in A549 cells. Here, high throughput multi-omics analysis identified the molecular effects of Zn intoxication on the proteome, metabolome, and transcriptome of A549 human NSCLC cells after 5 min to 24 h of Zn exposure. Multi-omics analysis combined with additional experimental evidence suggests Zn intoxication induces ferroptosis, an iron and lipid peroxidation-dependent programmed cell death, demonstrating the utility of multi-omics analysis to identify cellular response to intoxicants.
Asunto(s)
Ferroptosis/efectos de los fármacos , Pulmón/patología , Zinc/toxicidad , Células A549 , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Genómica , Humanos , NAD/biosíntesis , Necrosis , Unión Proteica/efectos de los fármacos , Factores de TiempoRESUMEN
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. This report details the automation, benchmarking, and application of a strategy for transcriptomic, proteomic, and metabolomic analyses from a common sample. The approach, sample preparation for multi-omics technologies (SPOT), provides equivalent performance to typical individual omic preparation methods but greatly enhances throughput and minimizes the resources required for multiomic experiments. SPOT was applied to a multiomics time course experiment for zinc-treated HL-60 cells. The data reveal Zn effects on NRF2 antioxidant and NFkappaB signaling. High-throughput approaches such as these are critical for the acquisition of temporally resolved, multicondition, large multiomic data sets such as those necessary to assess complex clinical and biological concerns. Ultimately, this type of approach will provide an expanded understanding of challenging scientific questions across many fields.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Metabolómica/métodos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteómica/métodos , Genómica/métodos , Células HL-60 , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Biología de Sistemas/métodos , Zinc/farmacologíaRESUMEN
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 10â¯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.
Asunto(s)
Células/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Redes y Vías Metabólicas , Apoptosis , Línea Celular , Supervivencia Celular , Cisplatino/farmacología , Biología Computacional/métodos , HumanosRESUMEN
Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining KM and kcat for various amino acid beta-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25-100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.