Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, ...especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."
Abstract
The pathogenesis of coronavirus disease 2019 (COVID-19) pneumonia remains poorly understood. The urine proteome of hospitalized patients with severe COVID-19 pneumonia, compared with severe ...non-COVID-19 pneumonia controls, was distinct and associated with lower abundance of several host proteins. Protein-specific machine learning analysis outlined biomarker combinations able to differentiate COVID-19 pneumonia from non-COVID-19 pneumonia controls.
Despite affecting up to 70% of HIV‐positive patients and being the leading cause of dementia in patients under 40 years, the molecular mechanisms involved in the onset of HIV‐associated ...neurocognitive disorders (HAND) are not well understood. To address this, we performed SILAC‐based quantitative proteomic analysis on HIV‐Tat treated SH‐SY5Y neuroblastoma cells. Isolated protein was fractionated by SDS‐PAGE and analyzed by nLC‐MS/MS on an Orbitrap Velos. Using MaxQuant, we identified and quantified 3077 unique protein groups, of which 407 were differentially regulated. After applying an additional standard deviation‐based cutoff, 29 of these were identified as highly significantly and stably dysregulated. GO term analysis shows dysregulation in both protein translation machinery as well as cytoskeletal regulation that have both been implicated in other dementias. In addition, several key cytoskeletal regulatory proteins such as ARHGEF17, the Rho GTPase, SHROOM3, and CMRP1 are downregulated. Together, these data demonstrate that HIV‐Tat can dysregulate neuronal cytoskeletal regulatory proteins that could lead to the major HAND clinical manifestation‐synapse loss.
Bacille Calmette-Guerin (BCG) vaccine can elicit good T
1 responses in neonates. We hypothesized that the pioneer gut microbiota affects vaccine T cell responses. Infants who are HIV exposed but ...uninfected (iHEU) display an altered immunity to vaccination. BCG-specific immune responses were analyzed at 7 weeks of age in iHEU, and responses were categorized as high or low.
subsp.
was enriched in the stools of high responders, while
was enriched in low responders at time of BCG vaccination. Neonatal germ-free or SPF mice orally gavaged with live
exhibited significantly higher BCG-specific T cells compared with pups gavaged with
and
differentially affected stool metabolome and colonic transcriptome. Human colonic epithelial cells stimulated with
induced a unique gene expression profile versus
. We thus identified a causal role of
in early-life antigen-specific immunity.
Objectives: The Fragile X Mental Retardation Protein (FMRP) is a widely expressed RNA-binding protein involved in translation regulation. Since the absence of FMRP leads to Fragile X Syndrome (FXS) ...and autism, FMRP has been extensively studied in brain. The functions of FMRP in peripheral organs and on metabolic homeostasis remain elusive; therefore, we sought to investigate the systemic consequences of its absence. Methods: Using metabolomics, in vivo metabolic phenotyping of the Fmr1-KO FXS mouse model and in vitro approaches, we show that the absence of FMRP induced a metabolic shift towards enhanced glucose tolerance and insulin sensitivity, reduced adiposity, and increased b-adrenergic-driven lipolysis and lipid utilization. Results: Combining proteomics and cellular assays, we highlight that FMRP loss increased hepatic protein synthesis and impacted pathways notably linked to lipid metabolism. Mapping metabolomic and proteomic phenotypes onto a signaling and metabolic network, we predicted that the coordinated metabolic response to FMRP loss was mediated by dysregulation in the abundances of specific hepatic proteins. We experimentally validated these predictions, demonstrating that the translational regulator FMRP associates with a subset of mRNAs involved in lipid metabolism. Finally, we highlight that FXS patients mirror metabolic variations observed in Fmr1-KO mice with reduced circulating glucose and insulin and increased free fatty acids. Conclusions: Loss of FMRP results in a widespread coordinated systemic response that notably involves upregulation of protein translation in the liver, increased utilization of lipids, and significant changes in metabolic homeostasis. Our study unravels metabolic phenotypes in FXS and further supports the importance of translational regulation in the homeostatic control of systemic metabolism. Crown
HIV-associated neurocognitive disorders (HAND) affect up to 70% of HIV positive individuals and are the leading cause of dementia in patients under 40 years. Despite this, the molecular mechanisms ...involved in the onset of HAND are not well understood. Among a number of plausible etiological agents of HAND, HIV-Tat has been shown to be neurotoxic in vitro and in vivo, but the basis of its induced neuronal dysregulation remains relatively poorly characterised, giving rise to various competing theories. This thesis describes differential, quantitative proteomic analyses of HIV-Tat-treated neuronal cells in vitro, the goal being to gain deeper insight into the underlying molecular basis of this HIV-Tat-mediated dysregulation, as well as to potentially inform better patient treatments in the future. To achieve this goal, deep, quantitative proteomic analysis of HIV-Tat treated SILAC-labelled SH-SY5Y neuroblastoma cells was carried out, alongside transcriptomic analysis of the same system in which 3077 proteins were identified and quantified with 407 proteins and 1074 genes being differentially expressed. Subsequently, label-free proteomics analysis was used to study the ability of lithium - a proposed new treatment for HAND - to suppress the HIV-Tat induced dysregulated molecular phenotype in SH-SY5Y cells in which 3757 were identified and quantified with 360 and 531 being significantly differentially expressed in HIV-Tat and HIV-Tat + lithium treated cells, respectively.
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