Support for mixed acquisition modes

Support for mixed acquisition modes Yishai  2017-11-21
We have some data running targeted analysis using the Lumos where we run EThCD PRM in parrallent to other modes (DDA MS3). We also run other mixed modes on the Lumos but are unable to load the data into Skyline. Do you plan on supporting such modes?

Brendan MacLean responded:  2017-12-09
Hi Yishai,
The first step would be to get us some example data with a Skyline document you wish you could load it into. You can do this by using File > Share - Complete on your document (to created a ZIP file) and then upload that and at least one raw data file to:

And let us know it is there. Then we will have a look and assess how hard it would be to get Skyline to achieve what you are looking for. If it is not super hard, then the answer to your question would be, yes, we plan on supporting such modes. If it is super hard, then we might still "plan on supporting such modes" but the plan would take longer to execute or have an indefinite time line.

But, help us have a look at what you would like to work. It might be easy to fix.

Thanks for reporting that this is not currently working for you.

Meital Kupervaser responded:  2018-10-14
Hi Brendan,

I uploaded a BSA digest we run in PRM that includes several modes of fragmentation and is not supported by Skyline at the moment. We use this for our QC of the Lumos and fragment using CID, HCD and ETD. At the moment we can only see the MS1 signal in Skyline, but not the MS2, which we would very much like to.
The file name is FS1LC2_BSA_HCD-IT_28082018_01.raw
We'd appreciate any help with that.

matt.chambers42 responded:  2019-01-10
Hi Yishai/Meital,

Sorry it's taken a while to get to this. I didn't have trouble getting the MS2 data to show up in Skyline after turning on MS2 quantitation. But what shows up doesn't make for coherent chromatograms because Skyline doesn't discriminate between activation types or analyzer types. You have 6 different scan modes going on in that file: FT-HCD, FT-CID, FT-ETD, IT-CID, IT-HCD, IT-ETD. Each mode has a different range of intensities, so they can't be combined in the same chromatogram. That means you need 6 different chromatograms, which seriously dilutes the number of data points per peak. Skyline itself can't yet do that separation, but if you use msconvert on the file 6 times you can separate out each mode:

msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer FT" --filter "activation cid" --outfile FS1LC2_BSA_HCD-IT_28082018_01-ft-cid.mzML
msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer FT" --filter "activation hcd" --outfile FS1LC2_BSA_HCD-IT_28082018_01-ft-hcd.mzML
msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer FT" --filter "activation etd" --outfile FS1LC2_BSA_HCD-IT_28082018_01-ft-etd.mzML
msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer IT" --filter "activation cid" --outfile FS1LC2_BSA_HCD-IT_28082018_01-it-cid.mzML
msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer IT" --filter "activation hcd" --outfile FS1LC2_BSA_HCD-IT_28082018_01-it-hcd.mzML
msconvert.exe FS1LC2_BSA_HCD-IT_28082018_01.raw --filter "peakPicking" -v -z --filter "analyzer IT" --filter "activation etd" --outfile FS1LC2_BSA_HCD-IT_28082018_01-it-etd.mzML

Then you can import these mzML files as "replicates" in Skyline and you'll get 6 chromatograms per transition, one for each mode. They will be on different tabs because they're being treated as replicates.

It is possible that Skyline could do the separation automatically straight from the RAW file, and make separate chromatograms for each analyzer/activation pair, but I'll let Brendan speak to when (or if) that feature might be added.
Brendan MacLean responded:  2019-01-10
This doesn't seem like an important enough use case to burden all Skyline users with more complex UI to support, nor is it clearly funded.

So long as it is possible, that will have to be good enough for now.

Thanks, Matt.