In bottom-up proteomics, a commonly observed amino acid modification is the oxidation of methionine (Met-O). I wonder if the observed Met-O reflect natural protein oxidation or if they rather stem from oxidation during sample preparation.
During sample preparation, several factors could influence the Met-O:
Typical sample preparation protocols include reduction of the sample by either DTT or TCEP. This step serves to reduce disulfide bridges. I wonder if this step would also lead to reduction of oxidized methionines. Still, this reduction step seems to be used even in studies that analyse natural protein oxidation (e.g. Walker et al., PNAS, 2019, Methods)
Sample preparation takes several hours at least, typically the digestion is even prolongued over night. Several modifications typically happen during sample preparation or during the measurement, e.g. deamidations. I did not find studies about the extend of Met-O happening during sample preparation. Klont et al. report differences between Met-O in four different sample preparation methods, but it is not clear where the differences stem from. They could either happen during sample preparation or the four methods have different capacities of detecting natural oxidations.
- Does the use of reducing agents modify oxidized methionines?
- Do methionine oxidations occur during LC-MS/MS sample preparation and measurement? How strong is this effect?
- Does anyone know a technique that discriminates native protein oxidations from those occuring during sample preparation? I found this publication, using 18O, but I cannot access it and thus cannot estimate if the protocol would be useful.