Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

Abstract

BackgroundIncreasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.FindingsHere we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.ConclusionsGiven both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

“…However, there is also precedent for Met oxidation causing large-scale changes in protein structure (Griffiths and Cooney 2002;Wolschner et al 2009;Pan et al 2010;Marondedze et al 2013). If this were the case, it might easily lead to exposure of a previously cryptic phosphorylation site or to hide a previously exposed site.…”

Relative Quantification of Sites of Peptide and Protein Modification Using Size Exclusion Chromatography Coupled with Electron Transfer Dissociation

“…Mass spectrometry has been successfully used to identify even labile protein modifications, including protein phosphorylation 1–2 , and O-GlcNAc modifications 3–4 , as well as identifying chemical modifications including covalent labeling for protein surface mapping 5–6 , crosslinking 7–8 , deamidation of asparagine 9–10 , and protein oxidation 11–12 . In all of these systems, the amount of modification that occurs at each amino acid of a peptide or protein can have serious consequences to the interpretation of the data, whether for understanding chemical or enzymatic modification mechanisms and consequences 13–16 or for interpreting data from covalent labeling or chemical cross-linking studies 7, 17–20 . However, when the same modification can occur on multiple amino acids in a peptide, quantifying what fraction of a given modification occurs at each potential modification site is a significant analytical challenge.…”

“…The partial separation of modification isomers often obtained by C18 reverse phase (RP) chromatography results in complicated data interpretation for both identifying and quantifying sites of modification. When all isomers can be separated to baseline, quantification should be possible by integration of the selected ion chromatogram for each peak (although inaccuracies using this method have been reported for oxidized peptides in model systems) 13, 17, 22–23 . When isomers perfectly co-elute, relative quantification is possible by MS/MS fragmentation of the isomeric mixture of modification sites at any reasonable point during the elution, with quantification by analysis of the relative intensities of each modified and unmodified product ion in the MS/MS spectrum 13, 24 .…”

“…When all isomers can be separated to baseline, quantification should be possible by integration of the selected ion chromatogram for each peak (although inaccuracies using this method have been reported for oxidized peptides in model systems) 13, 17, 22–23 . When isomers perfectly co-elute, relative quantification is possible by MS/MS fragmentation of the isomeric mixture of modification sites at any reasonable point during the elution, with quantification by analysis of the relative intensities of each modified and unmodified product ion in the MS/MS spectrum 13, 24 . The commonly used collision-induced dissociation (CID) fragmentation method has been shown to inaccurately measure the modification extent at residue level for several modification 25–27 , while electron transfer dissociation (ETD) has been shown to provide more accurate identification and quantification for several peptide modifications, including complex mixtures of peptide oxidation products 13, 24, 28–29 .…”

Proteomics of Important Food Crops in the Asia Oceania Region: Current Status and Future Perspectives

“…Also, new proteomics technologies and approaches such as the proteomic analyses of post-translational modifications 242 will significantly enhance our understanding of regulatory processes in plants. In addition, future studies will also afford new insight into palm defense mechanisms against such devastating pests as the red palm weevil Rhynchophorus ferrugineus 243 and may contribute to novel pest management strategies 244 .…”