Problem description

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:

  1. 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)

  2. 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.

My questions

  1. Does the use of reducing agents modify oxidized methionines?
  2. Do methionine oxidations occur during LC-MS/MS sample preparation and measurement? How strong is this effect?
  3. 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.

1 Answer 1


I know this was posted a long time ago and I'm not sure if you have found your answer but I am an author on two of the publications you andcite can offer some insight.

1.) Methionine oxidation, unlike cysteine oxidation, is irreversible without the action of a dedicated enzyme or catalyst. Neither TCEP nor DTT is able to reduce methionine sulfoxide. The only reducing agent that I know of that is strong enough to reduce methionine sulfoxide is MMA.

2.) The in-vitro oxidation of methionines during sample preparation is a well documented issue in LC-MS/MS analysis and it is known that the majority of in-vitro oxidation occurs during peptide digest as well as during electrospray ionization. The strength of the effect is highly variable and dependent on how long you digest proteins. For a typical overnight digest we observe effects that range from 0%-400%, depending on the peptide.

3.) The use of 18O labelled blocking reagents is the only currently available method that can prevent the accumulation of in-vitro oxidation. I'm the first author on the publication you cite and would be happy to send you a copy if you email me ([email protected]) I have also been working on improved methods using the same general strategy and would be happy to share those with you after publication

  • $\begingroup$ Hi John. Thanks for your reply. In the meantime, I found your paper on PMC. I will look into this. $\endgroup$
    – DaRealHonk
    Oct 7, 2020 at 19:51
  • $\begingroup$ could you please comment whether there was a study that directly showed that TCEP cannot reduce Met? $\endgroup$ Oct 12, 2021 at 19:50

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