Western blotting for post translational modifications vs. quantitative mass spectrometry: study of 3-nitrotyrosine

摘要

Western Blotting (WB) analysis is widely used to detect protein sequences that would specifically react with anantibody. Protein post-translational modifications (PTM) also can be detected by WB [1]. For example, anti-3-nitrotyrosine (3-NT) antibodies are generally commercially available. However, reliability of WB analysis for PTM detection remains doubtful as in the case of 3-nitrotyrosine detection in biological samples had led to a loss of WB signal while control nitrated proteins were added a biological matrix. Many theories occurred the most fascinating of which was a putative denitrase activity [2]. Here we investigated the influence of biological matrix, such as rat cardiac tissue homogenate, on the detection of 3-nitrotyrosine containing protein (rabbit muscle phosphorylase b, PhB, exposed to peroxynitrite in vitro).