Exclusion lists on a Q Exactive HF Orbitrap

[mnewmeyer]

I am attempting to add an exclusion list to an analysis I am running with a Q Exactive HF Orbitrap. I ran a series of samples with a Full MS-ddMS2 method that included polarity switching and generated an exclusion list from ions that are above my ddMS2 triggering threshold that are present throughout my run (i.e., ubiquitous elevated baseline) or that were present as defined peaks at specific retention times. As a result, some ions in the exclusion list do not have start and end times, so they are excluded for the entire run, and some ions have a retention time window. The total list, which includes both positive and negative mode ions, has >3,000 individual rows. Then, I reran my samples with this exclusion list added to my method. When reviewing the results, I see that the instrument is still triggering MS2 scans for ions that are on the exclusion list; however, some ions are being successfully excluded.

I verified that the "exclusion list" parameter on my method settings is turned on for both polarities. I have also noticed that for some compounds there is a co-eluting ion that is triggering the MS2, and the excluded ion is within the isolation window (already set pretty narrowly at 0.7 m/z) of the triggering ion; I can live with the fact that in these situations there is likely little I can do. However, there are many other cases where an excluded ion is eluting well within my retention time window and there are no co-eluting ions but an MS2 is still triggered. Mass differences between the observed ion and the mass in the list are well within 5 ppm.

Any help or suggestions would be greatly appreciated!

[GeorgeTFS]

Thanks for your question, @mnewmeyer. I will try to get you some feedback as soon as possible.

[mnewmeyer]

Thank you @georget! 

[FourierTFormer]

Interesting question @mnewmeyer! Are you calculating the 5 ppm within excluded mass directly from the scan header that triggered the relevant ms2 scan? For the exclusion list itself did you set both the m/z and charge to exclude or just the m/z itself (or maybe you used formula plus adduct)?  

[mnewmeyer]

To calculate the mass difference I looked at the mass in the MS1 spectrum in the scan immediately prior to the MS2 scan and compared it to the mass in the exclusion list. I did not indicate a charge state in the exclusion list itself, I only added an m/z value. Also, I did not indicate this in the original post but I also set the mass tolerance for the exclusion list to the instrument default (the value of which I cannot find anywhere).

[Kamel_GX]

Hello, did you manage to tackle the exclusion list issue? 

[mnewmeyer]

Hi @Kamel_GX! Unfortunately, I do not have a definitive resolution for this question yet. Some other projects have taken priority and I have not been able to dedicate a lot of time to this issue. I'm hoping that I can get back to it soon! 

[Kamel_GX]

Hello, thanks for prompt reply, how did you build the 3000 rows exclusion list? RT alignment of blank samples?

I think Thermo now has a software called AcquireX on Xcalibur 4.5 and above to do just that, building exclusion lists and optimize MS/MS analysis. Unfortunately, we don't have it

[mnewmeyer]

There were 2 types of interferences I wanted to account for:

1. ions that were present throughout the entire runtime (background ions, no specific peak)
2. ions with specific retention times

I collected data for a few blank injections and used Compound Discoverer to find ions with specific retention times (type 2 in the list above). For the ubiquitous background ions (Compound Discoverer won't find these) I converted the .raw file to an mzML format and used an R script to filter the data, and then combined the two lists.

My understanding of AcquireX is that it isn't supported by the Q Exactive HF, but maybe I'm mistaken. 

[Kamel_GX]

Yes, you are correct, it is not supported by the Q-Exactive HF or Q-Exactive plus in our case, I thought it was an offline data interpretation software but it is actually an online real time analysis, moves features back and forth between inclusion and exclusion lists. Thanks for all the info, will update you if I found out a solution to the issue.