Different m/z range for precursors and products

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Different m/z range for precursors and products user  2018-09-20
 

Dear Skyline team,

Does the m/z range in transition settings-instrument apply to both precursors and products? If I check “dynamic min product”, what will be the range for products? I have some PRM data from QE series, in which precursor m/z range is 400-1000 but product range is dynamic with minimum of 50.

Thanks,
Antony

 
 
Brendan MacLean responded:  2018-10-15

Answering a bit late, but it does apply to both, largely as a vestige of Skyline's start in SRM where they were the same, but also because now the most common reason they differ is in DIA, where the precursor range is usually defined by the DIA isolation scheme. Checking the "dynamic min product" is the right thing to use if you want Thermo dynamic minimum behavior for product ions.

But then the only way for you to force Skyline to pick only precursors from 400-1000 would be to define a temporary DIA isolation scheme with that range, and then later uncheck the "Auto-select all matching transitions" in the Transition Settings > Filter tab and then switch back to Targeted as you MS/MS acquisition method.

Hope this is helpful to someone, even if you, Antony, have moved on past this question.

--Brendan

 
user responded:  2018-10-16

Dear Brendan,

Nice to see your explanation. I did move on by setting a much wider m/z range to cover both precursors and products (unchecked “dynamic min product”).

I tested the method you suggested just now. I am afraid it does not work very well, since only 5 or 6 products are shown for many peptides in the setting as you suggested. I let Skyline to select the 10 most intense products based on library, and 10 products are shown for all peptides if I set a wide m/z range (see attached).

Kind regards,
Antony

 
Brendan MacLean responded:  2018-10-16

I don't think you could have possibly achieved what I was trying to suggest, because simply adding a DIA isolation scheme should only impact which precursors (peptide + charge state or labeling) Skyline chooses. It should have no impact on transitions in the precursors chosen at all. That was the point. You would leave the range in your Transition Settings - Instrument tab untouched, as wide as you want if transitions are picked from library spectra that come from the same instrument, as the library spectra will not contain any peaks outside the instrument range. Though, if you are starting from a library created with your instrument, neither should it contain any precursors from outside its detectable range. So, this whole discussion may be moot, unless you are actually trying to limit your target selection in a way that is different from how the library was collected.

However, if your settings change impacts the transitions inside the precursors at all, then you are not achieving what I have suggested. I am suggesting that you temporarily choose DIA as you MS/MS acquisition method and then set a custom isolation scheme which ranges from 400 to 1000, as the only way currently in Skyline to limit the range of precursors isolated to something narrower than the range of MS/MS spectra on your instrument.

If you still can't make this work, please include screenshots of what you have done to the settings as it will be easier to explain relative to those.

Great to hear that you otherwise mostly got the method you wanted already.

--Brendan