quantitation of protein carbonylation by dot blot

Yet, the specific role of specific protein carbonyls in the onset of particular biological impairments needs further investigations. From a medical perspective, protein carbonyls are highlighted as markers of protein oxidation, oxidative stress and disease. Though the implication of carbonylation on impaired protein functionality and poor protein digestibility is generally recognized, the underlying mechanism of such connections requires further clarification. From a practical perspective, the biological relevance of protein carbonyls in the field of food science and nutrition is still a topic of debate. From a methodological perspective, the routine dinitrophenylhydrazine (DNPH) method is criticized not only for the lack of accuracy and consistency but also authors typically perform a poor interpretation of DNPH results, which leads to misleading conclusions. In particular, protein carbonyls are involved in the formation of aberrant and dysfunctional protein aggregates, undergo further oxidation to yield carboxylic acids of biological relevance and establish interactions with other biomolecules such as oxidizing lipids and phytochemicals. The high reactivity of protein carbonyls in foods and other biological systems indicates the intricate chemistry of these species and urges further research to provide insight into these molecular mechanisms and pathways. Secondary protein carbonyls are introduced in proteins via covalent linkage of lipid carbonyls (i.e. α-aminoadipic and γ-glutamic semialdehydes) have been linked to radical-mediated oxidative stress, but recent studies emphasize the role alternative carbonylation pathways linked to the Maillard reaction. From a mechanistic perspective, primary protein carbonyls (i.e. Carbonylation is regarded as one of the most notable post-translational modifications in proteins and yet, this reaction and its consequences are poorly understood. All rights reserved.Protein oxidation is a topic of indisputable scientific interest given the impact of oxidized proteins on food quality and safety. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience.Ģ,4-Dinitrophenylhydrazine Aldehyde-reactive probe Biotin-hydrazide Fluorescein-5-thiosemicarbazide Protein carbonylation.Ĭopyright © 2015 Elsevier B.V. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins.

Quantitation of protein carbonylation by dot blot