• Sign In
  1. Requests

Ion Mobility of Fragments are "0"

support

View Request

view list print
Ion Mobility of Fragments are "0" Premy  2023-02-26
 

Hello,

I am importing and analyzing DIA-IM data using an Ion Mobility library and iRTs built from DDA-PASEF data. When clicking the apex of a peak, a purple IM extracted window is shown for the precursor but not for all fragments (with a label of IM = 0 above them). I'm a bit concerned that the fragments shown are not at the specific IM window but rather the entire IM range.

Is there a way to make sure the same IM extraction window is assigned to the fragments of a target?

Thanks,
Premy
 
 
Brian Pratt responded:  2023-02-27

May I have a look at the Skyline document and the mass spec result? That would be the fastest route to an answer.

Thanks for using the Skyline support board,

Brian Pratt
 
Premy responded:  2023-02-27

Hi Brian,

I've attached the shared file with a few examples with and without the ion mobility for the fragments.

Thank you for the help,
Premy
 
Brian Pratt responded:  2023-02-27

Looking at the IMSB file I can see that for some of the entries there's a high energy ion mobility offset that's the negative of the MS1 IM value.

Why that should be is the question - may I see the mass spec data D:\Users\Premy\20230206_DIA_windowtest\UltimateSplash_25da_std1_9_1_16153.d as well?

Thanks

Brian
 
Premy responded:  2023-02-27

Hi Brian,

Ah okay, I didn't check the offset value! The file is too large to attach here, what's the next best way to share this with you?

Premy
 
Brian Pratt responded:  2023-02-27

It's likely too large for an attachment - you can upload to http://skyline.ms/files.url though.

Brian
 
Premy responded:  2023-02-27

Hi Brian,

Got it, uploaded right now (same file name, but a zipped file).

Best,
Premy
 
Brian Pratt responded:  2023-02-27

Hmm, using just that file to create an IMSDB library I don't see the effect that's bothering us. Can you provide the entire set of replicates?

Thanks

Brian
 
Premy responded:  2023-02-27

Hi Brian,

I've uploaded three replicates for "UltimateSplash_25da_std1".

Additionally, could this be due to the fact that the IM library was built from DDA-PASEF data and is now being used to analyze DIA-IM data (although I didn't think this would be an issue)?

Best,
Premy
 
Brian Pratt responded:  2023-02-27

OK, I can reproduce the problem with the replicate UltimateSplash_25da_std1_9_1_16154. I will investigate and get back to you ASAP.

Brian
 
Premy responded:  2023-02-27

Hi Brian,

Thanks for all the support and help so far.

I had another related question I thought I would ask here as well. Is there a possibility for IM alignment across multiple runs? I sometimes see IM shifts across runs on different days, and it would be great to be able to adjust this!

Best,
Premy
 
Brian Pratt responded:  2023-02-27

In theory the instrument calibration takes care of that. Skyline just asks the Bruker data reader what IM to use given a CCS and charge, the calibration that's stored in the .d helps with that calculation but it's a black box to us. It sounds like maybe you need to be calibrating more frequently?
 
Brian Pratt responded:  2023-02-28

Thanks for the files, I can definitely use these to make improvements that will prevent this problem in the future.

For the moment, if you just edit that IM library in Skyline (use Settings>TransitionSettings>IonMobility>IonMobilityLibrary>EditCurrent) to remove the high energy offsets, when you reimport your data you should find everything working.

May I use some of your data for automated testing of this fix? Note that it will become (rather obscurely) public if we do use it.

Thanks

Brian Pratt