Dataset Information

OxSWATH applied to the redox characterization of secretomes under oxidative stress conditions

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. 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sandra Anjo

LAB HEAD: Bruno Manadas

PROVIDER: PXD006802 | Pride | 2019-02-15

REPOSITORIES: Pride

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Publications

oxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential proteomics screening.

Anjo Sandra I SI Melo Matilde N MN Loureiro Liliana R LR Sabala Lúcia L Castanheira Pedro P Grãos Mário M Manadas Bruno B

Redox biology 20190130

Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation without considering the variation in the total levels of the proteins. However, protein synthesis and protein degradation also belong to the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in PTMs-focused analyses, such as cysteine redox characterization. Therefore, a novel integrative approach combining the SWAT ...[more]

PMID: 30737169

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Dataset Information

OxSWATH applied to the redox characterization of secretomes under oxidative stress conditions

Publications

oxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential proteomics screening.

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