Dear Colleagues:
For those who haven't heard, I have retired after 40 years of serving the ABRF community. I will miss being involved with your chromatographic challenges. But before I go, there is this nagging question which I believe to be a misunderstanding of how sensitive proteomic separations are to ferric chelation, particularly when analyzing phosphorylated peptide digests.
The claim for the this data was that the sample contained C18 particles which were retained by the 0.2µm outlet frit of the Neo™ auto sampler. Remember the Neo does not have mobile phase passing through it so the sample is introduced to the RPC column in "~water" so only those peptides capable of eluting past the frit under those conditions will eventually be separated on the downstream RPC column.
No increase in pressure was observed, only the loss of signal halfway through the gradient indicating retention prior to entering the RPC column. So I ask you chromatographers: What would one expect if the frit contained iron and chelated hydrophobic peptides by their phosphate groups and not elute if the mobile phase in the injector was ~water? Wouldn't the chelated peptides act like a hydrophobic bonded phase and retain all further hydrophobic peptides? The peptides seen in the chromatogram would be those which could elute through the frit under the sample MP conditions (i.e. polar peptides which elute early on a C18 column under low organic conditions), but all more hydrophobic ones would not elute. Remember one sees a loss of signal at the MS interface if the needle is oxidized, so why not at the outlet frit of the injector if it contined ferric impurities?
To test this, try adding medronic acid (methylene diphosphonate) to the sample to cap the ferric sites or to displace the retained phosphopeptide to prove (or disprove) this hypothesis. It would be a great retirement gift to me if this clarifies our thinking so I an let this go now that I am retired.
With kind personal regards,
Amos Heckendorf
The Nest Group, Inc.
------------------------------
Amos Heckendorf
President
The Nest Group, Inc.
IPSWICH MA
------------------------------