Hi Lin,

thanks for your interest in the MRM-HR experiments and using Skyline for that also. I wrote that document you were looking at.

I also select 'rolling collision energies' as you indicate. I am using the CE equations as shown in the slide attached (those numbers originate from Christie Hunter). As I typically use a CES (collision energy spread of 3 or 4) that typically then works pretty well for my peptides (on the TripleTOF 5600).

You mention the MS2 fragment ion intensity is lower than the MS1 intensity. I see that also and that is to some degree in the nature of the experiment....

As I understand: The RT-scheduled MRM-HR experimental setup is as follows : as you have an inclusion list that the instrument then works off the instrument does not determine charge state in that particular experiment (as the precursor is not selected through data dependent assessment of the MS1 trace, but through the inclusion). That way in RT-scheduled MRM-HR setup (as described in the document you had referred to above) considers the precursor unknown charge state, it then selects from the collision energy table (my slide attached) what you had entered in as equation for unknown charge state (see in my case that is what one would use for charge state 2 (you can change that in the table).

If now your precursor in the MRM-HR list was a 3+ ion it will still use the equation defined for unknown (which in my case is optimized for 2+ ions). Typically that is not a problem as you have the CES that accounts for that ... I actually have not had any problems with this.

But if one is concerned you can either change the equation for unknown charge state or use the more classical way of setting up 'unscheduled MRM-HR) where you do not use the inclusion list but define specific MSMS experiments for preselected precursors in a loop (for a given cycle time) that then cycles through the entire gradient. I such experimental set up you can define the CE for each precursor individually and optimize specifically. But as then you are not scheduled the number of targets decreases quite a bit to maybe 20 or 30 peptides...

I think the fact that you see MS2 fragment ions at lower intensity, that is expected. However as it has such a much better selectivity (and less interferences) compared to MS1, in many cases this then still gives better quantitation in the end.
You can play with the equations a bit if you like to optimize etc but I typically just use the given equations (and together with the CES I typically get alright fragmentation that is good enough for quantitation, particularly also as it is full scan MSMS in MRM-HR, in the end you just use the best few fragment ions as you did not have to predefine those as one has to in MRM on the triple quadrupole).
Let me know any other questions or thoughts.

Birgit

Hi Birgit

Thank you for very detailed reply. It took me a while to find an available slot of 5600 to test different collision energy setting. I found our collision energy parameter for unknown charge state was set to use formula of singly charged. I compared the original setting (singly charged) and new setting (doubly charged) and found slightly improvement. For abundant protein, it is fine but I don't think it is good enough to give sufficient fragment to give confident quantification.
One thing occurred to me is that "dwell time". Correct me for the followings : In the case of sMRMHR, it is actually the time for collecting MS2. Since ABsciex averaged the spectrum, the "dwell time" should mainly affect the S/N ratio rather than intensity. However, I was still wondering if people in this discussion group have any idea about best "dwell time" for 5600?

thank you so much