I have trypsin-digested peptides from FACS-sorted samples, but they are contaminated with PEG.
I read in a paper that one of the solution for removing PEG is adding activated carbon to the PEG containing protein or peptide solution, and then removing the activated carbon by centrifugation or filtration.
Article SP2: Rapid and Automatable Contaminant Removal from Peptide ...
Alternatively go back over your sample preparation protocol and determine the source of the PEG contamination.
Hi Jose-Luis,
In my view (and also as Charles mentioned above), I would try to get rid of the PEG contamination by troubleshooting the source of the contamination and avoiding it from the beginning instead of doing an additional purification step which can also possibly impact the recovery of your peptides.
In my experience the mayority of PEG contaminations are coming from PEGylated detergents (like Triton X or NP40) used in a sample preparation step before LC-MS. Ther is a very nice description from Victoria Miller on how to avoid PEG contaminants in the following link
https://allumiqs.com/avoiding-peg-contaminants/
The only thing I would like to add to the description of Victoria is about using autoclaved tubes or tips. During autoclavation PEGs and other plasticizers can leach out of the platic remaining on the surface and getting rinsed into the samples when using this autoclaved tips or tubes. I personally never use autoclaved tips and tubes in any of sample preparation steps for this reason. However in case you are not sure you can rinse the tips or tubes with an organic solvent before using them.
Good Luck!
Murat
Thanks Leila Baharinikoo , Charles H Hocart , and Murat Eravci . We are trying to identify the source of contamination. It possibly originates from the FACS instrument. The samples were sorted and collected in regular 15 mL tubes, then ultracentrifuged, and the remaining volume was dehydrated using a vacuum centrifuge concentrator. After that, they were re-suspended in the same volume of 5% SDS, heated to 95ºC, and sonicated. Following this, they were transferred to low-binding tubes, and throughout the process, non-autoclavable tips were used and glass bottles for all the buffers. Finally, the S-Trap micro high recovery protocol was employed.
- Badges
- Science topic