|mProphet for DIA: Remove peptides to apply features & RT difference||d o debets||2019-06-19|
Dear Skyline team,
Recently I've performed a relatively big DIA experiment for the first time and I am experiencing some problems with applying the different features for the mProphet peak picking. I know some of these questions have been asked previously, but unfortunately I haven't been able to find an answer on this forum that could solve my isseus. I'd be very grateful if you could provide me with some insights!
First, some information about my experimental set up:
The first thing I noticed when looking at my model, is that it's not very good at separating my targets from decoys, but this is probably partly due to the fact that quite some important features were not used. First of all, I deleted all the peptides that didn't have a library dotp. These peptides made sense since in the majority of times there were not enough transitions matched to the library. However, there were quite a few of them and I had to remove them by hand, leading me to my first question: is there a smarter/more efficient way to remove these peptides?
After removal of these peptides, I looked at the peptides that had no information for the RT difference value feature. However there are loads of peptides that do not have this information, leading me to think that maybe something has gone wrong in the way that I set up my library/Skyline file. Leading me to my second question: Do you have any idea what could cause this? Maybe more generally, could you give me an idea why a peptide would not meet this criteria? And last but not least: how can I deal with this? There are way too many to delete them by hand, so I do not really know how to proceed from here.
As I said, it's the first time I am performing a DIA experiment, so some insights would be very much appreciated. I have attached the zipped Skyline file for you to have a look at if that's helpful. I have removed the majority of the raw files from the results there since otherwise the file would be way too big. Maybe leading me to my last question: is the file size that I have somewhat as expected? The output .skyd-file is around 380GB, which sounds like a lot to me?
Thanks a lot for your help in advance!