Proteome-wide analysis of the oxidation state of initiator methionines

In bacteria, protein translation is initiated with formylated methionine residues. A subset of initiator methionines are retained, whereas others are removed from nascent polypeptides. Initiator methionines are prone to oxidation and are readily converted to methionine sulfoxides (MetO) by reactive oxygen species. Oxidation of initiator methionines prevents their subsequent deformylation and hydrolysis, which can negatively impact the function of some proteins. To protect against deleterious methionine oxidation, cells can enzymatically reduce MetO residues using a conserved class of enzymes known as methionine sulfoxide reductases (Msrs). Analysis of the oxidation state of initiator methionines can, therefore, provide important information regarding their propensity for downstream processing that may influence the folding and stability of newly synthesized proteins. Here, we present two protocols, Methionine Oxidation by Blocking (MObB) and Methionine Oxidation by Blocking with Alkylation (MObBa), for quantitation of oxidation levels of methionines within complex protein mixtures using mass spectrometry-based proteomics. Among other applications, these two approaches enable researchers to investigate the interplay between the oxidation and processing of initiator methionines.