Settings questions arising from Webinar 21: PASEF

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Settings questions arising from Webinar 21: PASEF paga  2021-12-13
 

I've been going through the recently uploaded tutorial on DIA-PASEF, and I have the following doubts I would love to solve.

1.- I understand why they select only precursors with charges 2 and 3, but what I don't understand is why they decide not to look at the precursors (MS1 spectra). They decide to avoid using MS1-filtering, and decided to only use MS/MS filtering with a Centroid Precursor mass analyzer. Why would be a good idea to dispense on valuable precursor information which is used to determine transition quality (idotp)?

2.- On the same line, why is Centroid better than TOF for PASEF?

3.- When selecting MS/MS filtering with TOF Product mass analyzer, the default resolving power is 30.000. On the PASEF tutoriual, the chocen Centroid option was paird with a value of 30 for resolving power. What is the source of this huge difference? Is there a way I can check the resolving power of my DIA files in case I wasn't the one performing them?

Thanks a lot for the support, Skyline team!

 
 
Brendan MacLean responded:  2021-12-13

Hi Pablo,
This is Brendan, answering for the "they" to whom you refer. :-)

  1. I tried to clarify during the webinar that I was choosing more limited settings to reduce the processing cost, which already took 7 minutes in that webinar. Adding precursors would have made it take longer, maybe only 10 minutes total, but those minutes can seem long in a 50-minute demo. It also adds to the complexity of the processed data. So, it was just easier to present the MS/MS-only method, which works well, and in DIA we generally rely a lot more on the MS/MS signal than the MS1 signal. It is useful supporting information, however, and I fully support your using it in your own research.
  2. Bruker TOF data is so dense that they don't even really support our retrieving these spectra in profile mode. Bruker has always pushed us to used centroided spectra for their data. In the MacCoss lab, we have derived ourselves that Thermo centroided spectra work better for our data processing tha profile mode spectra. Only with SCIEX, is the centroiding algorithm not always beneficial. So, for SCIEX TripleTOF data we recommend you use the TOF setting.
  3. The resolving power of "30" to which you are referring is in the ion mobility dimension, where it is totally appropriate. The 30,000 resolving power you are referring to is in the m/z dimension. Remember, with this data you are applying filtering in both dimensions, and they have different resolving powers. Fortunately, Skyline does a very nice job of allowing you to visualize the filters applied in its Full-Scan view, as shown in the webinar and tutorial. Hopefully, this will help researchers increase their familiarity with how the filtering is applied in the IM dimension.

Thanks for your interest in using this exciting new functionality. I hope this reply is helpful to you.

--Brendan

 
paga responded:  2021-12-14

Hi Brendan,

Really appreciate your prompt and spot-on response, huge fan here!