DIA-MS Based Proteomics Combined with RNA-Seq Data to Unveil the Mitochondrial Dysfunction in Human Glioblastoma.

Mitochondrial dysfunctions underlie the pathogenesis in glioblastoma multiforme (GBM). Comprehensive proteomic profiling of mitochondria-specific changes in human GBM is still insufficient. This study carried out a DIA-MS based proteomic analysis on the mitochondria isolated from human primary GBM and peritumoral tissue (as paired control), and further compared those findings with the transcriptomic datasets. A total of 538 mitochondrion-specific proteins were rigorously confirmed, among which 190 differentially expressed proteins were identified. Co-regulations of the mitochondrial dysfunction pathway networks were observed, including significant up-regulations of mitochondrial translation and apoptosis, as well as down-regulations of OXPHOS and mitochondrial dynamics. Proteins related to FA, AA metabolism and ROS also showed significant variations. Most of these alterations were consistent in trend when compared the proteomics findings with the RNA-Seq datasets, while the changes at protein levels appeared to be more dramatic. Potentially key proteins in GBM were identified, including up-regulated pro-apoptotic protein CASP3, BAX, fatty acid oxidation enzymes CPT1A, CPT2, ACADM, serine-glycine enzymes SHMT2, GATM, ROS-related protein SOD2, GPX1, and CAT; and down-regulated dynamin-related protein MFN1, MFN2, OPA1, and OXPHOS components; and also several differentially expressed ALDH isoforms. This study systematically profiled the mitochondrial dysfunctions by combining proteomic findings and mRNA datasets, which would be a valuable resource to the community for further thorough analyses.

Proteomic characteristics of bronchoalveolar lavage fluid in critical COVID-19 patients.

Up to 10-20% of patients with coronavirus disease 2019 (COVID-19) develop a severe pulmonary disease due to immune dysfunction and cytokine dysregulation. However, the extracellular proteomic characteristics in respiratory tract of these critical COVID-19 patients still remain to be investigated. In the present study, we performed a quantitative proteomic analysis of the bronchoalveolar lavage fluid (BALF) from patients with critical COVID-19 and from non-COVID-19 controls. Our study identified 358 differentially expressed BALF proteins (P < 0.05), among which 41 were significantly changed after using the Benjamini-Hochberg correction (q < 0.05). The up-regulated signaling was found to be mainly involved in inflammatory signaling and response to oxidative stress. A series of increased extracellular factors including Tenascin-C (TNC), Mucin-1 (KL-6 or MUC1), Lipocalin-2 (LCN2), periostin (POSTN), Chitinase 3-like 1 (CHI3L1 or YKL40), and S100A12, and the antigens including lymphocyte antigen 6D/E48 antigen (LY6D), CD9 antigen, CD177 antigen, and prostate stem cell antigen (PSCA) were identified, among which the proinflammatory factors TNC and KL-6 were further validated in serum of another thirty-nine COVID-19 patients and healthy controls, showing high potentials of being biomarkers or therapeutic candidates for COVID-19. This BALF proteome associated with COVID-19 would also be a valuable resource for researches on anti-inflammatory medication and understanding the molecular mechanisms of host response. DATABASE: Proteomic raw data are available in ProteomeXchange (http://proteomecentral.proteomexchange.org) under the accession number PXD022085, and in iProX (www.iprox.org) under the accession number IPX0002429000.

Quantitative Proteomics Identified TTC4 as a TBK1 Interactor and a Positive Regulator of SeV-Induced Innate Immunity.

TBK1, STING, and MDA5 are important players within the antiviral innate immune response network. We mapped the interactome of endogenous TBK1, STING, and MDA5 by affinity enrichment MS in virally infected or uninfected THP-1 cells. Based on quantitative data of more than 2000 proteins and stringent statistical analysis, 58 proteins were identified as high-confidence interactors for at least one of three bait proteins. Our data indicated that TBK1 and MDA5 mostly interacted within preexisting protein networks, while STING interacted with different proteins with different viral infections. Functional analysis was performed on 17 interactors, and six were found to have functions in innate immune responses. We identified TTC4 as a TBK1 interactor and positive regulator of sendai virus-induced innate immunity.

A comparative proteomic analysis of Salmonella typhimurium under the regulation of the RstA/RstB and PhoP/PhoQ systems.

In pathogenic bacteria, the two-component regulatory systems (TCSs) play important roles in signal transduction and regulation of their pathogenesis. Here, we used quantitative proteomic methods to comparatively analyze functional networks under the control of the RstA/RstB system versus the PhoP/PhoQ system in Salmonella typhimurium. By comparing the proteomic profile from a wild-type strain to that from a ΔrstB strain or a ΔphoPQ strain under a condition known to activate these TCSs, we found that the levels of 159 proteins representing 6.92% of the 2297 proteins identified from the ΔrstB strain and 341 proteins representing 14.9% of the 2288 proteins identified from the ΔphoPQ strain were significantly changed, respectively. Bioinformatics analysis revealed that the RstA/RstB system and the PhoP/PhoQ system coordinated with regard to the regulation of specific proteins as well as metabolic processes. Our observations suggested that the regulatory networks controlled by the PhoP/PhoQ system were much more extensive than those by the RstA/RstB system, whereas the RstA/RstB system specifically regulated expression of the constituents participating in pyrimidine metabolism and iron acquisition. Additional results also suggested that the RstA/RstB system was required for regulation of Salmonella motility and invasion.

Protein turnover analysis in Salmonella Typhimurium during infection by dynamic SILAC, Topograph, and quantitative proteomics.

Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120, and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 and 99.30 min for the infection group and the DMEM group, respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding, and catalytic activity. The half lives of proteins involved in purine metabolism pathway were found to be significantly shortened during infection.

Analysis of Salmonella PhoP/PhoQ regulation by dimethyl-SRM-based quantitative proteomics.

SRM (selected reaction monitoring), a tandem mass spectrometry-based method characterized by high repeatability and accuracy, is an effective tool for the quantification of predetermined proteins. In this study, we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run. By applying this method to the Salmonella PhoP/PhoQ two-component system, we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg(2+) concentrations could be divided into two distinct patterns. For the time-course SRM experiment, we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns, providing a new clue regarding PhoP/PhoQ activation and regulation. Moreover, the results for iron homeostasis proteins in response to Mg(2+) concentrations revealed that the PhoP/PhoQ two-component system may serve as a repressor for iron uptake proteins. And ribosomal protein levels clearly showed a response to different Mg(2+) concentrations and to time.