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OxSWATH applied to in-vitro the characterization of redox state of recombinant DJ-1 proteins

ABSTRACT: Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. In this project it is presented the results from the validation of the method using the recombinant protein DJ-1.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sandra Anjo

LAB HEAD: Bruno Manadas

PROVIDER: PXD006803 | Pride | 2019-02-16

REPOSITORIES: Pride

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Project description:Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein, and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. This approach can be an important tool in redox centered approaches, being able to not only analyze the overall reduce/reversible oxidized state of proteins but able to be further applied in the characterization of the different oxoforms of the individual cysteines. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can be also performed, allowing a truly comprehensive evaluation of proteomics changes upon oxidative stimulus.

Project description:Glioblastoma (GBM), the most malignant primary brain tumor, is characterized by widespread heterogeneity, leading to poor and unpredictable clinical outcomes. Recent studies have provided evidences that the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. Here we investigate how GBM cells modulate glial cells, and how this modulation can influence back on the malignant phenotype of GBM cells. Primary mouse glial cultures were established and cultured in serum-free media (unprimed) or exposed to secretome derived from GL261 GBM cells (primed). Conditioned media (CM) from each glial culture were collected and a proteomic analysis was conducted. Glial cells CM (unprimed and primed) were also used in GL261 GBM cells to evaluate its impact in critical hallmarks of GBM cells, including viability, migration, and activation of tumor-related intracellular signaling pathways. The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells, consistent with a pattern of reactive astrogliosis. At the functional levels, CM derived from unprimed glial cells favored an increase in cell migration capacity, while CM from primed glial cells was more efficient in promoting GBM cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Together, our results demonstrate that glial cells can have a different impact on the progression of GBM tumors, suggesting that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells. Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

Project description:Fresh fish are highly perishable food products and their short shelf-life limits their commercial exploitation, leads to waste and has a negative impact on aquaculture sustainability. New non-thermal food processing methods, such as High pressure (HP), are being investigated to prolong shelf-life while assuring high food quality. We applied several tools to evaluate the impacts of HP processing on European sea bass (Dicentrarchus labrax) fillets quality and shelf life. The data here presented includes visual and physical measurements of flesh quality and the microbiome and proteome profiles of control and HP-processed sea bass fillets (600MPa, 25ºC, 5min), after isothermal storage (2°C) for different periods ranging from 1 to 67 days. Color (L-, a- and b- values) change and texture (hardness, cohesiveness and adhesiveness) parameters were obtained by using appropriate colorimeter and texture analyser, respectively, during refrigerated storage. Bacterial diversity was analysed by Illumina high-throughput sequencing of the 16S rRNA gene in five pooled DNAs from control or HP-processed fillets after 1, 11 or 67 days and the raw reads were deposited in the NCBI-SRA database with accession number PRJNA517618. In addition, high-throughput sequencing of the internal transcribed spacer (ITS) region targeting yeast and moulds was run for control or HP-processed fillets at the end of storage (11 or 67 days, respectively), being deposited under SRA accession PRJNA517779. Quantitative label-free proteomics profiles were analysed by SWATH-MS (Sequential Windowed data independent Acquisition of the Total High-resolution-Mass Spectra) in myofibrillar or sarcoplasmic enriched protein extracts pooled for control or HP-processed filets after short (1d) or long-term (11-67 days) storage. These data support the findings reported in “High pressure processing of European sea bass (Dicentrarchus labrax) fillets and tools for flesh quality and shelf life monitoring” (Tsironi et al. 2019).

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OxSWATH applied to in-vitro the characterization of redox state of recombinant DJ-1 proteins

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