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Liquid chromatography coupled to mass spectrometry (LC-MS) is one of the most powerful tools in identifying and quantitating molecular species. Decreasing column diameter from the millimeter to micrometer scale is now a well-developed method which allows for sample limited analysis. Specific fabrication of capillary columns is required for proper implementation and optimization when working in the nanoflow regime. Coupling the capillary column to the mass spectrometer for electrospray ionization (ESI) requires reduction of the subsequent emitter tip. Reduction of column diameter to capillary scale can produce improved chromatographic efficiency and the reduction of emitter tip size increased sensitivity of the electrospray process. This improved sensitivity and ionization efficiency is valuable in analysis of precious biological samples where analytes vary in size, ion affinity, and concentration. In this review we will discuss common approaches and challenges in implementing nLC-MS methods and how the advantages can be leveraged to investigate a wide range of biomolecules.
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Espectrometría de Masa por Ionización de Electrospray , Cromatografía LiquidaRESUMEN
The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC-MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/ß signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1ß, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host-pathogen interactions.
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As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP-N-acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms. Graphical abstract.
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Adenosina Trifosfato/metabolismo , Metabolómica , Microfluídica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adenosina Difosfato/metabolismo , AutomatizaciónRESUMEN
Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells, resulting in reversal of Treg suppression. Importantly, TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression, which may be used as a strategy to advance tumor immunotherapy.
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Neoplasias de la Mama , Glucosa/metabolismo , Melanoma , Linfocitos T Reguladores/inmunología , Receptor Toll-Like 8/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Senescencia Celular , Femenino , Humanos , Inmunoterapia , Melanoma/metabolismo , Melanoma/terapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microambiente TumoralRESUMEN
Sample pretreatment in conventional cellular metabolomics entails rigorous lysis and extraction steps which increase the duration as well as limit the consistency of these experiments. We report a biomimetic cell culture microfluidic device (MFD) which is coupled with an automated system for rapid, reproducible cell lysis using a combination of electrical and chemical mechanisms. In-channel microelectrodes were created using facile fabrication methods, enabling the application of electric fields up to 1000 V cm(-1). Using this platform, average lysing times were 7.12 s and 3.03 s for chips with no electric fields and electric fields above 200 V cm(-1), respectively. Overall, the electroporation MFDs yielded a â¼10-fold improvement in lysing time over standard chemical approaches. Detection of multiple intracellular nucleotides and energy metabolites in MFD lysates was demonstrated using two different MS platforms. This work will allow for the integrated culture, automated lysis, and metabolic analysis of cells in an MFD which doubles as a biomimetic model of the vasculature.