Hi,
I am performing a purification of an complex of unknown constitution by following the enzyme activity, and one of the final steps is performing an Ion Exchange Chromatography. Now I want to perform mass spectrometry on the samples, however the fractions are rich in salts and diltued (500uL). I need somehow to concentrate the sample in order to submit the samples.
Any recomendations?
Why this fraction collected from the IC is "rich in salts" ? the ions should be easily separated from organic compounds in IEx (?)
You can use SPE to enrich your analytes and get rid of the salts.
After the SPE you will collect the compounds of interest in organic solvent, which can be evaporated to a desired volume.
regards,
G
Hi, Grzegorz, thanks for your response.
Well, it is rich in salts since I elute the proteins of interests at around 500 mM salt in a volume of 500 uL per fractions, meaning that e.g. direct trypsinization for the MS is not possible. That's why I need to concentrate the proteins and get rid of the salts that would kill the trypsin.
Concentrate the protein using a centrifugal ultrafilter. Reduce the salt content by rediluting the protein with buffer lacking salt. Finally, reconcentrate it. All steps take place in the same filter. Make sure the molecular weight cutoff of the filter is smaller than the molecular weight of the protein. Usually, 10,000 Da cutoff is adequate.
For proteins to remove salt you can use GFC (hydrophilic GPC/SEC).
regards
G
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