Question about method setting of data dependent NL-MS3

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Hi everyone,

I’m a user of Thermo Fisher LTQ-Oribitrap Elite.

Recently, I have some problems of method setting in data-dependent neutral loss MS3 (DD-NL-MS3) scan mode.

Since my research candidates are all have a great characteristic that is consecutive loss of two different moiety (R1 and R2) on their structure by CID in MS.

So that these two specific neutral loss mass (R1 and R2) can be used as feature for screening these candidates.

Firstly, we test a single compound that would consecutively neutral lose R1 and R2 moiety by CID.

We setting parent mass of this compound and turned off dynamic exclusion function to evaluate the method.

In our results, we found a lot of MS3 events were triggered for this compound, but they all came from same fragment ion (loss of R2).

However, the other fragment ion (loss of R1) can not be triggered the MS3 event since their intensity is always weak than other one (loss of R2).

The mass error and intensity were all satisfy data dependent criteria.

Detailed result and method parameters can be see as follows links.

(MS2 fragmentation spectrum)
https://drive.google.com/open?id=1yHHcf ... XDBWUmgFrT

(Chromatogram of MS1, MS2 and MS3)
https://drive.google.com/open?id=1oOAyZ ... DuHuZoPfBf

(Method parameters)
https://drive.google.com/open?id=18sErG ... IldPC724aE

Our ultimate goal is to see all candidates precursor that can be triggered MS3 scan twice for loss of R1 and R2, separately.

I’ll appreciate any comments and help.

Thank you all.
since both losses show up in MS2, could you just do standard data-dependent MS2 on everything, and create neutral loss chromatograms for the two losses, looking to see where both losses coincide? This can only occur where a compound elutes that is capable of both losses R1 and R2. The only advance you achieve by the MS3 method is that you could try to look for compounds that must lose R1 and R2 in the correct order.

I notice that R2 is exactly two of R1; I'm not sure what basis you have for believing that R2 is only possible after R1 has fallen off? The MS2 spectrum for your positive control would tend to suggest that either loss is possible from the outset. Low-energy CID does allow all sorts of internal rearrangements of molecules.

The more complexity you put into a data-dependent MS^n method, the more risk there is that it will fail to trigger on a valid peak for obscure reasons. It's probably safer to keep the method simple and mine the data than make the method complex, relying on looking where the method chose to trigger.
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