Identifying Carbonylated Proteins in Brain Tissue

The article, which has just be accpeted by the American Chemical Society, is entitled 'Proteomic approaches to identifying carbonylated proteins in brain tissue' and is freely available online at: http://pubs.acs.org/doi/pdfplus/10.1021/pr101014e

Abstract

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using non-treated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis, and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were useful in identifying carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.

American Chemical Society

Proteomic approaches to identifying carbonylated proteins in brain tissue
María Linaresa,b, Patricia Marín-Garcíaa,b,c, Darío Méndeza,b,¶ , Antonio Puyeta,b, Amalia Dieza,b and José M. Bautistaa,b,*