DIA TIC does not match DDA Mark Athanason  2020-08-14 08:07
 

Hi again Brendan,

I know this is not a skyline question, but since I followed your tutorial for setting up the DDA and DIA methods in Xcalibur I figured I would ask here. In the past this worked perfectly well with a 2 hour gradient, but now I'm running the two methods as a one hour gradient and something funky is going on with the DIA acquisition. The DIA and DDA chromatography parameters are exactly the same, and the DIA instrument method is as shown in your tutorial. I have attached the two method files along with a screen shot of the TICs to demonstrate what I am talking about. If you can see something I'm obviously doing wrong, I would be very appreciative. This is being run on a nanoAquity LC with a picochip spray source coupled to a Thermo QE HF mass spec.

Best
Mark
 
 
Nick Shulman responded:  2020-08-14 12:22
If you would like, you can send us your two .raw files and we might be able to see what is going on.

You can upload large files here:
https://skyline.ms/files.url

-- Nick
 
Mark Athanason responded:  2020-08-14 14:38
Hi Nick,

Thanks a lot for being willing to take a look. I uploaded a compressed file called "N_DDA_1.zip" that contains the DDA and DIA run. These are injections of the same sample.

Best
Mark
 
Nick Shulman responded:  2020-08-14 16:34
How many DIA runs do you have that look like that?
I think that the chromatography is the same between your DDA and DIA run, but, for some reason, there are a thousand times fewer ions inside of the mass spectrometer during those periods where the DIA TIC looks flat.
-- Nick
 
Mark Athanason responded:  2020-08-16 08:40
Nick,

I was re running this sample set hence my delayed response. This is just a trial run, so it's a small experiment. I have 4 samples, I'm injecting them thrice to build the DDA library and once for the DIA acquisition. All 4 raw files (3 DDA 1 DIA) are injecting from the same vial. The DDA runs look fine, then the DIA run looks like a blank.

This method worked perfectly fine in the past. This was with a two hour gradient, though. All I did was shorten up the times. Since it looks like the sample is not being injected for the DIA run (even though it's definitely taking something from the vial) somehow it's getting lost between the needle and the analytical column.

Mark