Session: Session 3

Mass Spectrometric Profiling of Methionine Oxidation During Actin Filament Formation

Actin filaments are crucial components of the cytoskeleton, providing structural integrity and mechanical properties to cells. The dynamic nature of actin filaments (F-actin) and the reversible polymerization of actin allow cells to use actin monomers (G-actin) for various functions, including cytokinesis, cell movement, organelle transport and the formation of cell protrusions. This transition between G-actin (soluble monomer) and F-actin (component of insoluble polymer microfilaments) is regulated through redox-dependent reversible oxidation at two specific methionine residues: M-46 and M-48. This redox regulation is mediated by the opposing actions of Mical and MsrB1 proteins. Measuring the oxidation levels of these 2 specific methionine residues can enhance our understanding of several biological processes.

In proteomics, it is well-documented that sample processing and mass spectrometry can introduce artifactual methionine oxidation, making it challenging to distinguish between in vivo methionine sulfoxides and those formed artifactually during the analysis. To accurately assess methionine oxidation, a method to « freeze » the methionine state in biological sample is required. Recently, a selective blocking method using alkylation, termed MObBA, was developed. This method employs iodoacetamide at an acidic pH to selectively modify methionine, resulting in two distinct methionine forms: in vivo methionine sulfoxide and in vitro alkylated methionine.

                Using HeLa cells and purified actin, combined with Mical and MsrB1 proteins, we evaluated this new selective blocking by alkylation methodology to measure methionine oxidation at both MS1 and MS2 levels. This approach allows for accurate differentiation between methionine sulfoxides formed in vivo and those introduced during proteomic analysis, providing more precise insights into the redox regulation of actin dynamics.