Oxidation and reduction of amino acid side chains in proteins is a normal part of redox regulation in cells where slight surges in reactive oxygen species (ROS) are generally dealt with by oxidation of suflhydryl groups to mixed disulfides. After an oxidative stress (OS) episode has passed, these disulfides are reduced back to sulfhydryls and the normal redox potential of the cell is restored. Collectively, proteins can be oxidized in more than 35 ways. All of these post-translational modifications occur in three basic ways that are distinguishable by mass spectrometry. One involves oxidative cleavages in either the protein backbone or amino acid side chains in which Pro, Arg, Lys, Thr, Glu or Asp residues are most likely to undergo oxidative cleavage. A second mechanism is by indirect addition of lipid oxidation products such as 4-hydroxy-2-noneal, 2- propenal or malondialdehyde to proteins. Mass increases with this type of modification and is unique to the appended group. Mass spectrometry is an analytical technique that measures the mass of molecules an atoms. The ionization of peptides for mass spectrometry is typically carried out using either electrospray ionization (ESI) or matrix assisted l aser desorbsion ionization (MALDI).Recent advances in mass spectrometry analytical techniques have already provided more accurate and more quantitative ways to measure oxidative modifications in the cell. The emerging branch of proteomics, so-called ‘redox proteomics’, is not only determined simply by protein levels but also by post-translational modifications to proteins, which might be significant, particularly in cells undergoing an oxidative stress.

Hi Narendra, the short answer is yes, there are different methods available to identify whether a protein is reduced vs oxidized using mass spectrometry. Most methods are indirect and rely on differential labeling of reduced vs oxidized cysteines. Exampels are initial irreversible alkylation of reduced cysteines using e.g. iodoacetic acid, iodoacetamide or N-ethylmaleimide, then reducing the oxidized cysteines and reacting those with a second alkylating reagent. This can be either completely different from the first alkylator, or a heavy-isotope labeled version of the first alkylator to be able to separate them in the MS. Other versions include direct irreversible labeling of sulfenic acids with dimedone, or using the ICAT approach or different versions of iodo-TMT probes.

A very important step/problem is high background oxidation signal, and you will have to make sure to properly degass your buffers and use of fresh reagents to make sure you are efficiently alkylating your samples.

When you say you have your protein in mixture, do you mean that you have it in a complex mixture, such as a cell lysate?

Yes, I am talking about cell lysate and I am interested in one specific proteins.

The approach will of course also depend on the type of MS you can or intend to do, and the type of oxidation that you expect (whether it is on cysteine or on a different amino acid), sulfenic acid/dimer/irreversible oxidation/nitrosylation/etc.

Is there a specific reason that you want to apply MS, like absolute quantification of identification of which cysteine is oxidized? If your looking at one specific protein then gel/western blot-based methods might be an alternative.