We use Acetone precipitation to concentrate /clean-up proteins from urine samples. However, it's difficult to fully resuspend samples post precipitation. We've tried many different solubilization buffers including additives such as SDS or Urea but always get left with a cloudy solution which contains significant amount of protein (checked by running pellets on SDS-PAGE). Has anyone experienced this issue?
It might be easier to deal with the samples if you don't use acetone precipitation. Try concentrating the urine with ultrafiltration instead.
Hi Stoyan, I agree with Adam. Urine has serum albumin and other proteins in it that will denature with acetone treatment. Once denatured, many of these proteins will never be solubilized.
Try using non-denaturing ultrafiltration instead.
Thank you all for the valuable suggestions.
Aravinth, will reduce the starting volume to try get a smaller pellet. Would like to also stay away from detergents since these will have to be removed prior MS. Any suggestions on non detergent containing buffers?
Fatemah, what buffer did you use for resuspending the pellets?
Pavel, we also make sure pellets don't completely dry out. This has helped solubilizing pellets from other sources but in this case no improvement. We've also tried a range of other precipitation methods but outcome was no better.
Adam, the reason we stayed away from ultrafiltration up to now is because we have a relatively large sample numbers to deal with (over 200) and was looking at the simplest sample processing methods in order to improve throughput. Given that it's difficult resuspending post precipitation (and may have to use detergents which would have to be removed adding further steps) will give ultrafiltration a go. What size filters would you suggest, perhaps 3K, since as far as I know urine contains many peptides /small proteins that may be of interest?
Steingirmur, do you think the fact that Albumin does not resupend may be an advantage since this may act as a "depletion" step or would it be too unspecific? I suppose will never know what else is being lost that may be of interest and perhaps will have reproducibility issues?
Stoyan, Have you considered freeze-drying your samples? That will, of course, concentrate solutes other than proteins, which may interfere with your analysis. On the other hand, it allows many samples to be concentrated simultaneously.
Hi Stoyan, albumin binds many peptides. If you precipitate albumin out of the mixture, you might also lose whatever is bound to albumin.
http://scholar.google.com/scholar?q=peptides+bind+serum+albumin&btnG=&hl=en&as_sdt=1%2C21
No, but seems like a very good way to get around the large sample numbers. Will give this a go. We using NEST C18 plates post digestion to desalt prior MS which should be sufficient to remove contaminants. So as long as they don't interfere with trypsin it should be OK. My only other concern would be rgd protein degradation due to bacteria etc present in the samples. Do you think freeze drying will be sufficient to "sterelize" the samples or should add some anti-bacterial agents post resuspending?
Urine should be sterile to begin with if was collected into sterile containers, unless the donor had a urinary tract infection. Freeze-drying should kill off any bacteria, I would think. If the dry samples are resuspended under sterile conditions, they should be OK. Otherwise, you could add 0.02% sodium azide as a preservative.
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