I am performing mass spectrometry on a Co-IP sample from mouse brain to identify native protein partners of my protein of interest. I struggling with PEGs contamination in samples which cover part of my peptides peaks. I’m using Dynabeads M-270 Epoxy from Thermo. I perform the entire procedure according to the manufacturer protocol for direct MS analysis. To elution I’m using HPH EB buffer (0.5M NH4ON, 0.5 mM EDTA), all plastics came from Eppendorf. Do you recommend any other elution strategy which can omit PEGs contamination problem?
Dear Piotr,
we encountered the same problem with my colleague. The same samples prepared by him were always contaminated while mine were w/o PEG - using same workspace, same cells, same labware. Conclusion - this was person-related. The difference between us - I am almost bald while he was extensively using hair grease (or whatever this is called in english). And this contains the PEG... Hopefully this gives you ideas as where to look for possible contamination.
Hi Piotr,
The best way to purify such samples and make your life easy with downstream mass spectrometry analysis, is to purify the complexes using SDS-PAGE followed by GeLC-MS/MS.
The SDS--PAGE step will remove all the additives. I usually run a very short gel (to stack the proteins in a small acrtylamide area) and use commercial gels which have very short (couple of mm) stacking gel below the wells.
After brief staining (I use colloidal Commassie stain which is rapid and does not contain acetic acid in stain/destain steps), excise the gel section and proceed with in -gel proteolysis. The peptides generated have to be cleaned up. I use hand packed Stage Tips (also commercially available from ThermoFisher). The final peptide pools are dried to a droplet, reconstituted in 0.1% formic acid and introduced to LC-MS/MS.
Using the above approach, I have never encountered contaminating peaks from plastics or PEG.
Good luck,
Hediye.
- Badges
- Science method