|Questions on baseline intensity and peak calling||lparsons||2019-05-07|
Briefly, our process starts by running two samples in triplicate MS1-only scans. We now use OpenMS for feature detection and consensus mapping to produce a list of peaks of interest that are selected as a result of group t-tests (3 runs of sample A vs 3 runs sample B). These features are then used to produce an inclusion list for the instrument to then do a directed DDA where features are pre-selected for MSMS. The MSMS data then is run through MaxQuant, and the MaxQuant data is fed into Skyline. In Skyline we then omit the raw MSMS data, instead using the raw MS1-only data in its place so that the peak intensities from the MS1-only data will be used for the MSMS features.
The results of this generally look pretty good. However, there are some features that come out of this with vastly different intensities from Skyline than what they had in OpenMS. In particular, there are some features where we know that sample B should not show any intensity while sample A should. On some of these samples, Skyline reports very low intensity (exported as "Total Area MS1" in Peptide Quantification report) where we expect none and yet on others Skyline reports very high intensities (up to 10^6 or more) when we expect none.
This leads me to a couple questions
I also looked at the dot products of these features to see if that could lend some insight. When I plot median dot product vs median B intensity - for the peaks where I expect to see zero intensity from B but am getting positive "Total Area MS1" for B - I don't see a strong correlation though there is a wide distribution of median Total Area MS1 in the range of median dot products > .085 and < 1.