Hello! I am running an experiment involving biotin labelled DNA oligonucleotides. I hope to identify nuclear proteins bound specifically to my ~70bp oligonucleotides and elute the nuclear proteins using µMACS Miltenyi System. I will subject these proteins for in solution trypsin digestion followed by C18 spin column and mass spectrometry. Hence, I need to avoid the use of high salts (interfere with trypsin digestion) and detergents (interfere with mass spectrometry).
I am at odds on how I can separate my proteins from the 50nm microbeads. I have six options currently:
(1) Elute from the column with 5-50% acetonitrile gradient. I will speedvac before in solution digestion. This leaves the microbeads in the column and selectively elutes the protein. However I am concerned that acetonitrile might cause the proteins to precipitate in the column.
(2) Elute from the column with 2M ammonium bicarbonate. This leaves the microbeads in the column and selectively elutes the protein. However I am concerned that 2M is not strong enough to elute my proteins, and I would have to dilute to 50mM for in solution trypsin digestion.
(3) Elute from the column with HFIP. But I think HFIP is a very harsh organic solvent and might denature the proteins.
(4) Spin down the microbeads after reduction step. I cannot seem to find any protocol including spinning down microbeads, but I did see a paper that claims that 50nm microbeads are too small and non-sedimenting.
(5) Re-run MACS after reduction and collect the flow through. However, I might potentially lose many proteins.
(6) Just send the magnetic beads for mass spectrometry. Is this possible? Wouldn't the magnetic beads clog the C18 column.
I would really really appreciate any help you can provide as to which option sounds most feasible. Thank you so much!
I would do an on-bead digestion with trypsin, which will give you the peptides that can then be followed by cleanup (if needed) and MS. This is quite a common approach for elution and any mass spec service should be able to advise you on it.
In addition to suggestions regarding directly sending the magnetic beads to mass spec analysis for on-bead digestion, You may use buffer exchange to make the in-solution digest condition as MS-compatible and bead free.
Assuming you are using the streptavidin-coated magnetic bead in your µMACS Miltenyi Strategy for isolating the biotinylated DNA thus conjugated proteins. Elute the biotinylated compound as the procedure suggested (I would recommend using free biotin which works under mild and non-denaturation conditions; The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer)...Other harsh conditions may take place in this stage. Whatsoever, an eluted fraction may be subjected to dialysis, UF, or a desalting column to get rid of elution composition. If you use certain detergents, FASP would be useful for both detergent elimination (chaotropic dissociation of the large micelles followed by filter elimination) and also for protein digestion on membrane digestion...If you need to use detergent for elution MS-compatible detergents may also be tested but I am not sure about their elution efficiencies (not tested personally)...There are many other alternatives as abovementioned...
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