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Topic: Protecting -SH groups (Read 6536 times) previous topic - next topic

Protecting -SH groups

Hi everyone,

In our experiments we often see -SH compounds (such as reduced glutathione - GSH) disappear and form disulfide bonds (GSSG in the case of glutathione).
To prepare our samples we use 50% methanol extraction, followed by 10-kDa cut-off filter to remove proteins, followed by vacuum evaporation.
I am wondering if other researchers experience this problem and how they deal with it. Do you add any buffers to your extraction medium?



Re: Protecting -SH groups

Reply #1
Both GSH and GSSG are present in cells.
But during the sample preparation, once cells are broken, GSH is exposed to air and  oxidized into GSSG very quickly.
Adding reducing agent into sample converts GSSG into GSH, then only GSH will be detected. Therefore, I don’t think there is a good way in sample preparation for these “active” molecules, any metabolomics results on these active molecules should be careful.

if you are only interested in GSH/GSSG, you can use a fluorescent assay to measure GSH/GSSG. You only need minimal sample preparation for assay measurement.

Re: Protecting -SH groups

Reply #2
I realize this topic is old, but the only way to preserve the redox state of the biological material for MS is to trap the reduced thiols with an alkylating agent. Different groups have employed NEM or IAM for this while the sample is still aqueous, or after recovering the sample from acidified conditions (low pH will lead to protonation for the overwhelming majority thiol groups, especially free glutathione and Cys). This procedure would modify all accessible thiol groups at relatively similar rates, varying predominantly based on the sulfur pKa (it gets more complicated if you care about protein Cys -- denaturation is crucial for non-selective labeling).

I have not seen a method to do this sort of procedure built into a larger untargeted platform, but multiple methods that utilize LC-MS quantification of multiple glutathione and/or Cys species after thiol alkylation have been published within the last decade.

One consideration with any alkylating reagent will be side reactions (applies moreso to IAM) and modifications of the adducted species (applies to mostly to NEM). Some have also observed that IAM will degrade to iodine in storage leading to artifactual reduction in some cases.

Lastly, I will add that I see clear evidence of artifactual oxidation of glutathione in acetonitrile preparations, but it is nowhere near as complete as it would be in aqueous conditions under the same extract handling.