Friday, February 18, 2022

Tutorial --the easy way to set up pasefPRMs!


We just had to go through this in our weekly proteomics group meeting so it is fresh in my mind. I'll move fast before it fades. This way of doing pasefPRM is exclusively for the TIMSControl version above. 

Even if you just got the ACS paper that came out 2 weeks ago on pasefPRM, if your TIMS control has been updated since ASMS2021 (Halloween 2021) you'll find things are slightly different. 

You can build your target list exclusively through Skyline if you want. Go right ahead. That's awesome. 

OR you can get your TIMSControl updated, process some DDA data through MSFragger or MaxQuant and then just build your targeted list like you would on another family of instruments you might be more familiar with. Here's how I do it with MSFragger.

I'm going to dig through a K562 digest for examples (click to expand) but I basically need my 1/k0, my observed peptide m/z charge and my retention time in seconds. I get these values from the PSM.tsv file from FragPipe generated results. 

And this is why you want the newest TIMSControl: 

You get a target editor! You can punch all these in, or you can make a CSV file and import it (way faster).

To do this first Export this empty target list. Now you get a CSV file that contains the headers exactly as they should be. 

Open that in Excel (just remember to only save it as a CSV, not an Excel) now enter your values from your MSFragger results.
You do need a range for your ion mobility so make a column in my MSFragger results to the left and right that are 1/k0 -0.05 and 1/k0 + 0.05 copy those columns into my CSV. 
Once you've saved that as a CSV, import that -- then generate your schedule. 

Obviously this is a very simple example I just made up, but you get these two visualizations that help you know if you're staying within the effective capabilities of the instrument. I purposely stacked two peptides at 32 minutes with different IMS ramps (highlighted in orange) just to show what that looks like. The image will scale as you load more targets and will provide a pretty intuitive visual way of seeing that is happening at each point in your experiment. There is obviously an upper limit for what you can target as you move across ion mobility ramps and optimizing that vs the MSinterval will be something you'll have to think about as you increase the number of targets, but otherwise you're good to go! 

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