Does anyone have any suggestions for the use of an alternative method for reducing a protein then performing LCMS analysis?
In our experiments we focus on a single protein to perform HDX studies and usually those proteins that are heavily disulfide bonded are reduced using TCEP because it was found to be more compatible than beta mercaptoethanol on the LC. Although the specific protein I am studying is a very stubborn in reducing one of it's domains. Any other reducing agents or methods someone may suggest? I may have to do an on offline digestion but the amount of protein I have is limited and would like to avoid any additional steps.
I would advise DTT as well. 25mM DTT pH 8.6 solution at 80ºC along one hour may be a starting trial condition for testing your material. Good lucky!
No not yet i was thinking about that. We usually have a range of conditions we reduce in that includes GuHCl and pH 2.7 so I think it should still hopefully work.
Depends on what you want to do, but we use 6 M urea, 50 mM Tris-HCl pH 8.8 with 2% DTT 15 min RT, then acetone precipitate the protein and resuspend in 6M Urea, 50 mM Tris-HCl pH 8.8 with 2.5% iodoacetamide 15 min RT - works well. Do not go above RT!
Usually I did 5 mM DTT in 10 mM Ammonium Bicarbonate, 60 min at 80 degrees celsius
TCEP has more advantage in reducing proteins, it will reduce protein in wide range of pH where the reformation will not happen in the wide range of pH as well the reducing rate is far better than DTT.
so its a try where change the pH using buffers with TCEP as reducing agent and check out the uncleaved domain
DTT is the better one i this case. I liked the whole discussion ad it was equally useful to me.Thanks for all the contributors.
Even though reforming of disulfide bonds is less likely with TCEP, it can happen in some conditions. Therefore, reduced samples should be used immediately or alkylated.
Thanks for all these suggestions, just got time to comment. We cannot use high temp to increase efficiency of reduction because deuterium exchange labeling is pH and temperature sensitive. All the reduction has to be done at low temp (usually we reduce on ice) and at low pH (2.7). This is the challenge of doing deuterium exchange experiments on proteins that are heavily disulfide bonded. The end goal for these experiments is to determine the best denaturing and reduction conditions that will produce numerous fragments of the protein during online proteolysis which is why reducing as much as we can at conditions of low temp and pH is optimum.
Filter Aided Sample preparation method would be the best to denature (Urea followed by SDS) ,reduce and modify proteins prior to Mass spectrometry with minimum loss.
For HDX, my suggestion is to using UREA and DTT to fully denature and reduce the protein. By using this method, you have to use trap column to remove those reagents before load to the analytical column. Based on some experiments, protein without denature is hard to be reduced by DTT. Please play with the UREA concentration in your quenching buffer to denature your protein first.
I agree that denaturation with urea is the best approach. However, you should be extremely careful in performing this experiment so that you do not generate artefactual carbamylation of your protein. I have seen large data sets where 10-15% of the peptides are carbamylated from the sample prep step. Watch reaction temperature and pH, and it's probably best to use a fresh urea solution.
We make what's called "quench stocks composed of XM GuHCl, .8% formic acid and XmM TCEP right now. The results we get with the frozen stocks are reproducible in terms of digestion fragmentation of a single protein. The denaturation is done at RT and pH 2.7 so everything is exact because the labeling has to stay quenched during deuteration. Although I will try searching for peptides now with carbalymlation and see i if i get anything further.
You can reduce your protein at higher pH with DTT and then you can protect your thiol groups with iodoacetamide. After that you can simply exchange the slightly alkaline buffer for an acidic one, let's say, by ultrafiltration or something similar. Maybe that would work for your HDX experiment.
Best of luck
Thanks for the suggestion but I'm afraid the label will come off when you increase the pH. Low pH is necessary to keep the exchange reaction 'quenched". I have been thinking of keeping the TCEP and then using hydrostatic pressure to increase the breakage such as the instrument that Pressure Biosciences has. May have to demo it to try i though.
I want to analyse EPO in biological samples.
Can anyone help me with a trusted procedure using maiia kit and LCMS for tryptic digest identification?
I need a detailed proc. because i tried once and failed
Regards...
- Badges
- Science topic
- Similar topics
- Mathematics
- Statistics