We are working on the LC-MS analysis of intact membrane proteins by using reverse phase HPLC or UPLC columns for online separation. We have issues with protein recovery and column life time for large membrane proteins with current setup. Would someone can share their experience with the column selection or methods? Many thanks in advance!
Perhaps HILIC-columns are a good option, since they have a rather polar stationary phase, while not requiring to use apolar solvents.
Hello!
To separate proteins with a reverse phase column and measured with an LC / MS system has always been a problem. Basically, the protein is always through the organic fraction (eg acetonitrile) of the mobile phase or is denatured at the latest in the ESI source.
It used to be like using a C4 column. Today I would rather use a monolithic column (eg ProSwift RP) or a column with an extremely large pore size (eg MabPac RP).
But especially in ESI ion traps you will not be able to measure very large proteins. The limit at ESI ion traps is approximately at about 60 kDa. In the routine has the best results with ESI ion trap at about 30 KDa.
Best regards
Burghard Cordes
Hao, people usually use C4 for small proteins but I have no idea how you can move it through in a native stage.
Dear Hao,
Actually, I always had scarce results in whole hydrophobic protein mixture RP-HPLC separation but please, find attached a technical note about this topic. I hope this can help as a point to start for the difficult task.
All the best,
Francesco
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