Blot-MS of Carbonylated Proteins: A Tool to Identify Oxidized Proteins.
The efficiency of proteostasis regulation declines during aging and the failure of protein homeostasis is common in age-related diseases. Protein oxidation is a major contributor to the loss of proteome homeostasis, also called "proteostasis," precluding protein misfolding and aggregation. So, the identification of the molecular pathways impaired by protein oxidation will increase the understanding of proteostasis and the pathophysiological conditions related to the loss of proteostasis. Sample derivatization with dinitrophenyl hydrazine and western blot immunoassay detection of carbonylated proteins (commonly known as Oxyblot™) coupled to mass spectrometry (blot-MS) is an attractive methodological approach to identify proteins that are more prone to carbonylation, a typical oxidative modification of amino acid residues. The integration of blot-MS data of carbonylated proteins with bioinformatics tools allows the identification of the biological processes more affected by protein oxidation and that, eventually, result in the loss of proteostasis.In this chapter, we describe a blot-MS methodology to identify the proteins more prone to oxidation in biological samples, as cell and tissue extracts, and biofluids. Analysis of mitochondria isolated from cardiac tissue is provided as an example. Bioinformatic strategy to deal with data retrieved from blot-MS experiments are proposed for the identification of relevant biological processes modulated by oxidative stress stimuli.
Department of Chemistry, Mass Spectrometry Centre, QOPNA, University of Aveiro, Aveiro, 3810-193, Portugal.
Rita
R
Ferreira
Department of Chemistry, Mass Spectrometry Centre, QOPNA, University of Aveiro, Aveiro, 3810-193, Portugal.
Pedro
P
Domingues
Department of Chemistry, Mass Spectrometry Centre, QOPNA, University of Aveiro, Aveiro, 3810-193, Portugal.
Francisco
F
Amado
Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto, Porto, Portugal. rvitorino@ua.pt.|Department of Medical Sciences, iBiMED-Institute for Biomedicine, University of Aveiro, Aveiro, Portugal. rvitorino@ua.pt.|PortugalQOPNA, Centro de Espectrometria de Massa, Departamento de Química, Universidade de Aveiro, Aveiro, Portugal. rvitorino@ua.pt.
Rui
R
Vitorino
D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D050050:Protein Carbonylation / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism*
D020543:Proteome
Immunodetection; Mass spectrometry; Mitochondria; Oxidative posttranslational modifications; Proteomics; Two-dimensional gel electrophoresis