Retention time alignment applied to MS2 trace?

[michaelwitting]

Hi,

after having the previous issues resolved (more or less), I'm working on the next level of my SWATH workflow. On single file level it is working quite good at the moment, but in the next step I would like to load a whole batch of files to process them in parallel.
I was wondering if the alignment in the MS1 trace also affects the MS2 traces? To work with a batch of files I have to make sure that the MS2 data is shifted the same way as the MS1 level, otherwise the correlation analysis I'm using for deconvolution won't work.

Best regards,

Michael

[jorainer]

Very good point! I'll check that and (if it doesn't) fix it.

[jorainer]

Since I've never had SWATH data in my hands I would need your help @michaelwitting on some basic questions:

1. do you do (chromatographic) peak detection only on MS1 or also on MS2?
2. retention time correction: you do that only on MS1 and apply the same correction to all MS levels?
3. correspondence: how is that expected to work? Group peaks separately for each MS level?

Might be that I have to change some basic concepts in xcms based on the stuff above.

[michaelwitting]

My current workflow idea looks like this:

1. Peak pick and align in MS1 Level (Alignment should also affect MS2, so that it is shifted the same way as MS1)
2. Split into MS1 and MS2 Level
3. Split MS2 Level into the different SWATH pockets according to precursor mass
4. perform Peak picking in MS2
5. search for each MS1 Peak in respective SWATH pocket for fragment candidates
6. get EICs MS1 peak and all fragment candidates
7. correlate MS1 and MS2 EICs
8. Reconstruct MS2 spectrum based on correlation values
9. Export MS2 spectrum as .mgf file

My plan is to reconstruct a MS2 spectrum for each peak in each individual file. Like this I can make sure that each spectrum is deconvoluted correct. Afterwards they can be merged, if it turns out that they are identical.

So regarding your questions:

1. Yes, peak detection in performed in both Levels. But MS2 Peak detection should be performed after alignment
2. RT correction on MS1 is sufficient, as long as the MS2 level from each file is shifted the same way.
3. Not needed. MS1 alignment and RT correction is sufficient

[jorainer]

Thanks @michaelwitting for the detailed answer.
One points that come immediately to my mind is that subsetting operations usually drop adjusted retention times. I'll have to fix that or allow for special cases.

[jorainer]

I think that should be closed by now. Feel free to reopen if needed.