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Scheduled PRM Global Peptide standard issue

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Scheduled PRM Global Peptide standard issue sivapira  2018-06-07 13:50
 
Hello Skyline Support Team,

I'm working on a PRM experiment where I'm planning to use BSA as global standard (spiking BSA to sample before digestion). I have run a preliminary experiment with an unscheduled method and I have refined the method. Now I'm ready to run a scheduled method but it seems like I have to do at least 3 scheduled injection due to the cycle time limit (I'm using a QExactive where a max 39 precursors can be monitored simultaneously).

My question is do I include the peptides for BSA for all 3 scheduled injections and if yes how do I standardize the peptides from Injection #1 with BSA peptides from Injection #1?

Please correct me if my thought processes are wrong and thank you for the help.

Regards,
Guru
 
 
Nick Shulman responded:  2018-06-07 14:08
Yes, you should collect data for these global standards in all of your injections.

When you are doing global standard normalization, Skyline uses the peak area of the standard from the same result file as the analyte.
 
Brendan MacLean responded:  2018-06-07 15:09
If you mark the peptides as Global Standards in Skyline and then use one of the Skyline operations (File > Export > Isolation List or Method) to produce your instrument method, Skyline will automatically include those targets (and any targets marked as standards: Global, QC or iRT) in your methods. If you use some other method, like using Skyline reports (as described in the recent tutorial webinar on PRM - http://skyline.ms/webinar17.url) then you will need to ensure yourself that the standards are in each method.

Thanks for asking your question on the support board.

--Brendan
 
sivapira responded:  2018-06-08 12:32
Thank you Nick and Brendan for the help.