Can anybody tell me the energies in eV used in Orbitrap HCD for proteomics? I would like to know how much bigger they are compared to CID and how much smaller they are compared to real HCD. Thank you!
Christian,
the "higher energy" in HCD refers to the higher radiofrequency voltage that is applied to retain most fragment ions in the C-trap. So, the higher energy is not needed to induce fragmentation but to keep the fragmented ions in the C-trap. According to the original literature, Makarov et al. raised the RF up to 2500 V (vs. 1500V in previously "normal" operation, ie. before HCD became available). This is not something that most users have access to change.
This is not to be confused with the collisional energy that is applied to induce fragmentation in the first place. That obviously depends on the mass, charge and nature of the collision gas. This should be similar to what is used in CID. Depending on the application, we use mostly somewhere between 35-50eV.
I would recommend this paper for additional information:
http://www.nature.com/nmeth/journal/v4/n9/full/nmeth1060.html
Hi Sonja,
Thank you for your excellent answer and the link!
Christian
Hi Christian, it is actually difficult to obtain an accurate number on the energies even from Thermo, who came up with0 "HCD", which I believe is largely a marketing term.
I did a direct comparison with MS/MS of peptides and phosphopeptides obtained on Q TRAP instruments after the publication came out, and with regard to the fragment ion types and even relative intensities results came out remarkably similar - virtually superimposable.
From a results point of view I have filed HCD under "beam-type CID", which is same or similar as what you obtain on QqToF and QTRAP instruments, so my guess (but not knowledge) is that the energies applied are also quite similar.
Hi Christof, I agree and find the term somewhat confusing/misleading. Thank you for your contribution - I got a similar impression when comparing QqTOF CID and Orbi HCD data. I like your designation "beam-type CID".
P.S. Did not know that you moved to an MPI.
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