Chromatography Forum

Dear colleagues,

I try to enrich phosphorylated peptides (or proteins) prior to mass spectrometry. I have so far tried an IMAC HPLC column kindly provided by Dionex and the ProteoExtract Phosphopeptide Capture Kit (also IMAC from CALBIOCHEM). Both these approaches seemed to work, @ least with standard substances, however, only when using rather large concentrations (such as 40pmol absolute entering the respective process).

Any experience in this field and thus any “helpful kicksâ€

The column you received from Dionex was a beta product, and so were the operating instructions. Would you please send me as much detail as you have liberty to disclose on what you were doing and the results? I am planning to do more internal testing on the phosphopeptide application later this month, and I would greatly benefit from your comments. I will update you with the results. You may email me directly at mark.tracy@dionex.com and please cc patrick.mccarthy@dionex.com

SMC,

You can work with smaller amounts but that requires a lot of custom work (i.e. bring everything down to the capillary level, pack your own columns etc) and optimization of a lot of steps.

I do not work directly on that project so I can not comment more on that.