Dear Experts:
We know that only when a peptide is fragmented only once meaning that a precursor is breaked only once to form two parts that the b y ions nomenclature can be true.
But I think the fragment ions can go on to collide with the inert gas atoms resulting in the fragment ions or product ions being fragmented again to form a secondary fragmentation. But infact, the ratio of secondary fragmentation is low and the proteomics building will collapse if a fragment ion can be framented again and again.
It seems the charge state,length of the precursor, the intramolecular rearrangement and the energy state of precursor can be the explanation for this question but there is no details from what I have found on some resouces.
Any one can give me some materials explaining this? or just give me a explanation to help the novice. Thank you very much.
If we start from given mode of fragmentation only. Firstly it depends on the amount of collision energy you introduce to the system. Bonds breaking needs energy. Secondly, charge state. For the fragment to be detected in MS it has to carry a charge. For that reason, the higher charge state of the precursor is, the bigger is the chance to get charged fragments (simplifying a bit).
@Valeriia Kuzyk Hi，Valeriia Kuzyk，thank you for your answer. Do you mean that if we enhance the collision energy then there will be more secondary fragmentation? The higher the CE,the more charges the precursors take, the bigger chances the fragments will be fragmented again?
I asked my self the same question multiple times... It's hard to understand how gas phase charged driven and radical driven processes can took place.
I might suggest this paper:
Gas-phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools
journal of mass spectrometry Vol 43, Issue 10 Oct 2008 pp 1301–1319
In principle, you can see multiple levels of fragmentation depending on the activation time and upon collision energy that you are using. You can experiment with this yourself a little bit to get a feel for it. Depending on the instrument you are using, you can measure an energy resolved set of experiments where you ramp up the collision energy and monitor the intensity of the fragments as a function of the collision energy. Keep in mind that fragmentation is a statistical process, where different fragmentation channels may open up at different internal energies. Another thing to think about here is the timescale of unimolecular dissociation and how this relates to the collision timescale.
Another experiment you can do, if your instrument supports it, is to look at MS3. Select a fragment from your mass spectrum, and fragment the fragment. You will likely see that many of the fragments produced by this shortened peptide sequence are also present in your parent CID spectrum. So in reality it can be difficult to know where a fragment comes from. However, in many applications of CID for proteins (those are not interested in fragmentation mechanism, but with sequence), it is the ability to identify fragments and ultimately sequence that matters.
There are techniques where secondary fragmentation is expected and observed. For example, spectroscopic techniques such as IRMPD or VIDPD, where energy is added to the system via photon absorption by a chromophore. As long as the chromophore remains intact in a fragment, you will be able to see further absorption and fragmentation of daughter species.
I totally agree with Steven Daly:
"There are techniques where secondary ... techniques such as IRMPD or VIDPD, where energy is added to the system via photon absorption by a chromophore. Omissis."
In the last three years the number of works, posters and talks regarding VIDPD and IRMPD at the ASMS has grown exponentially!
Thank you very much @Steven Daly and @Andrea Carra, I almost understand the mechanism.
In fact, recenty when I was using Maxquant to identify the proteins I can found many secondary fragment in the MS2 spectra which were marker by Maxquant. But these are omitted by the search engine and only the b y series ions are used in the peptides indenfication. May be that's the reason why we need to optimize the CE and dwell time so that we can maximize the percentage of b y ions in all frament ions.
I will follow your advice trying find the secondary profile,that is really interesting.
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