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|oxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential proteomics screening
|Anjo, Sandra I.
Melo, Matilde N.
Loureiro, Liliana R.
|This work was financed by the European Regional Development Fund (ERDF) through the COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under projects: PTDC/NEU-NMC/0205/2012, POCI-01-0145-FEDER-007440 (ref.; UID/NEU/04539/2013), POCI-01-0145-FEDER-016428 (ref.: SAICTPAC/0010/2015), POCI-01-0145-FEDER-016795 (ref.: PTDC/ NEU-SCC/7051/2014), POCI-01-0145-FEDER-029311 (ref.: PTDC/ BTM-TEC/29311/2017), POCI-01-0145-FEDER-30943 (ref.: PTDC/ MEC-PSQ/30943/2017) and PTDC/MED-NEU/27946/2017; and by The National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125 (ref.: ROTEIRO/0028/2013). S.I.A, was supported by Ph.D. fellowship SFRH/BD/81495/2011, co-financed by the European Social Fund (ESF) through the POCH – Programa Operacional do Capital Humano and national funds via FCT.
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|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 SWATH-MS method with differential alkylation using a combination of commonly available alkylating reagents (oxSWATH) is presented, by which it is possible to integrate the information regarding relative cysteine oxidation with the analysis of the total protein levels in a cost-effective high-throughput approach. The proposed method was tested using a redox-regulated protein and further applied to a comparative analysis of secretomes obtained from cells cultured under control or oxidative stress conditions to strengthen the importance of considering the overall proteome changes. Using the OxSWATH method it was possible to determine both the relative proportion of reduced and reversible oxidized oxoforms, as well as the total levels of each oxoform by taking into consideration the total levels of the protein. Therefore, using OxSWATH the comparative analyses can be performed at two different levels by considering the relative proportion or the total levels at both peptide and protein level. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can also be performed, allowing a truly comprehensive evaluation of proteomics changes upon the oxidative stimulus. Data are available via ProteomeXchange and SWATHAtlas with the identifiers PXD006802, PXD006802, and PASS01210.
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|FMUC Medicina - Artigos em Revistas Internacionais
I&D CNC - Artigos em Revistas Internacionais
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