Project description:Suzuki-Miyauara cross-coupling has been used for mass-spectrometry-based chemoproteomics, including for target deconvolution and site of labeling studies. We also demonstrated that Suzuki-Miyauara cross coupling and CuAAC can be combined for dual target labeling for mass spectrometry-based proteomics. Furthermore, multiplexed CuAAC Suzuki-Miyaura chemoproteomics platform has been successfully applied to identify the protein targets of bifunctional cysteine- and lysine-reactive crosslinking probes.
Project description:We utilized the well-characterized murine T cell transfer model of colitis to find specific alterations in the intestinal luminal proteome associated with inflammation. Mass spectrometry proteomic analysis of colonic samples permitted the identification of ~10,000-12,000 unique peptides that corresponded to 5610 protein clusters identified across three groups, including the colitic Rag1 -/- T cell recipients, isogenic Rag1 -/- controls, as well as wild-type mice. Bioinformatic analyses on host and microbial proteins found specific proteins and GO term functionalities unique to each group, as well as GO terms shared across the three cohorts. We further demonstrated that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia that was substantiated with 16S rDNA sequencing.
Project description:Recent studies demonstrated that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts induced augmented glycolysis and production of alpha smooth muscle actin (αSMA) and collagen1. Knockout of COUP-TFII decreased glycolysis and collagen1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.
Project description:Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across the two mouse cohorts, as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control; thereby, limiting genetic and environmental variables that influence microbiome composition.
Project description:Established bacterial proteome sample preparations, including sonication and bead beating, leave insoluble carbohydrate-rich cell envelope pellets with an abundance of vital proteins often overlooked or missed in LC-MS/MS analyses. Triflic acid selectively removes glycans and we demonstrate that in comparison to sonication alone, incubation of whole bacterial cells as well as post-sonication insoluble pellets yields membrane and cell envelope-associated proteins for LC-MS/MS detection. We provide a detailed side-by-side comparison of triflic acid and sonication preparations for Gram- (Pseudomonas aeruginosa), Gram+ (Bacillus subtilis), and a complex bacterial human distal gut microbiome sample. Further, human Jurkat cells that lack a peptidoglycan and are readily solubilized by established methods, reveal only subtle differences in measurable proteins by LC-MS/MS between sonication and triflic acid preparations. Critically, we show that our new triflic acid-based proteome preparation method is broadly applicable and greatly improves our ability to detect and quantitate bacterial cell envelope proteins.
Project description:We performed quantitative proteomics of 60 human-derived breast cancer cell lines to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the two omics datasets to identify and characterize the subtypes of breast cancer and showed that they conform with known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled molecular feature datasets. The datasets are made freely available as a public resource on the LINCS portal. We anticipate that these datasets, either in isolation or combination with measurements of complementary molecular features, can be mined for the purpose of predicting drug response, informing context in mathematical models of signaling pathways, inferring cell-type or subtype specific pathways activities of unperturbed cellular states, and identifying markers of sensitivity or resistance to therapeutics.
Project description:We performed quantitative proteomics and phosphoproteomics of 43 human-derived breast cancer cell lines to a depth of 11, 000 proteins and 45,000 phosphopeptides respectively. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the two omics datasets to identify and characterize the subtypes of breast cancer and showed that they conform with known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled molecular feature datasets. The datasets are made freely available as a public resource on the LINCS portal. We anticipate that these datasets, either in isolation or combination with measurements of complementary molecular features, can be mined for the purpose of predicting drug response, informing context in mathematical models of signalling pathways, inferring cell-type or subtype specific pathways activities of unperturbed cellular states, and identifying markers of sensitivity or resistance to therapeutics.
Project description:Background: The developing field of osteoimmunology supports importance of an interferon(IFN) response pathway in osteoblasts. Clarifying osteoblast-IFN interactions is important because IFN is used as salvage anti-tumor therapy but systemic toxicity is high with variable clinical results. In addition, osteoblast response to systemic bursts and disruptions of IFN pathways induced by viral infection may influence bone remodeling. ZIKA virus(ZIKV) infection impacts bone development in humans and IFN response in vitro. Consistently, initial evidence of permissivity to ZIKV has been reported in human osteoblasts. Hypothesis: Osteoblast-like Saos-2 cells are permissive to ZIKV and responsive to IFN. Methods: Multiple approaches were used to assess whether Saos-2 cells are permissive to ZIKV infection and exhibit IFN-mediated ZIKV suppression. Proteomic methods were used to evaluate impact of ZIKV and IFN on Saos-2 cells. Results: Evidence is presented confirming Saos-2 cells are permissive to ZIKV and support IFN-mediated suppression of ZIKV. ZIKV and IFN differentially impact the Saos-2 proteome. Both ZIKV and IFN suppress proteins associated with microcephaly/pseudo-TORCH syndrome (BI1, KIF20 and UBP18), and ZIKV induces potential entry factor PLVAP. Conclusions: Transient ZIKV infection influences osteoimmune state, and IFN and ZIKV activate distinct proteomes in Saos-2 cells, which could inform therapeutic, engineered, disruptions.
Project description:Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We finally demonstrate how measurements of reactivity in native versus denatured proteomes can further discriminate essential cysteines amendable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.
Project description:Proteomics has revealed that the ~20,000 human genes engender a far greater number of proteins, or proteoforms, that are diversified in large part by post-translational modifications (PTMs). How such PTMs affect protein structure and function is an active area of research but remains technically challenging to assess on a proteome-wide scale. Here, we describe a chemical proteomic method to quantitatively relate serine/threonine phosphorylation to changes in the reactivity of cysteine residues, a parameter that can affect the potential for cysteines to be post-translationally modified or engaged by covalent drugs. Leveraging the extensive high-stoichiometry phosphorylation occurring in mitotic cells, we discover numerous cysteines that exhibit phosphorylation-dependent changes in reactivity on diverse proteins enriched in cell cycle regulatory pathways. The discovery of bidirectional changes in cysteine reactivity often occurring in proximity to serine/threonine phosphorylation events points to the broad impact of phosphorylation on the chemical reactivity of proteins and the future potential to create small-molecule probes that differentially target PTM-modified proteoforms.