Missing MS2 from Mass Inclusion List.

Missing MS2 from Mass Inclusion List. mwmann  2019-10-10

Hi, much like everyone else here, I should probably mention how incredibly useful Skyline has been for me. I've been using it for the last 2.5 years, starting as a learning tool for MS and now using it for my own projects as a graduate student. That being said, I would probably still consider myself a beginner with this software. Right now I'm using Skyline to help me quantify changes in histone ptms, and it's not an understatement to say that I couldn't do it without this software. However, I'm running into a weird issue related to MS2 spectra not being recognized in Skyline.

I'm running Skyline Daily on Windows 10.

I'll start by detailing my workflow:

  1. I run my samples on a bruker QTOF using a DDA workflow. I've manually added certain masses to a mass preference list so that some ions would fragment at all points during their elution. This is to facilitate quantification of co-eluting isobaric ions. (I'll admit, it's possible that this step isn't working as intended, but it wouldn't explain all the behavior I'm seeing).
  2. I convert my files to centroided .mzml files and use Metamorpheus to calibrate and run a search for modified peptides. I import the resulting .mzid files into skyline as a spectral library with a cutoff of 0.99. My peptide settings allow a large number of PTMs (10), but restrict peptides to those found in the library.
  3. I import a fasta of selected histones (just H3 for now), and remove repeated peptides.
  4. I import my .mzml files and use the peptide identification times on each spectra to select peaks.

All of this seems to work well. However, when I look at MS2 spectra, I notice that I have relatively few MS2 peaks, even when peptide ids are shown on the spectra. I've attached an image demonstrating this. I can click on the IDs and see my fragment ions changing, but Skyline doesn't seem to have any MS2 at those timepoints. On the image, you can see the highest scoring ion as a straight line from practically zero abundance to its max, That same ion in the ID spectra increases, but not in a straight line manner.

Noting another poster had a similar issue which was resolved by changing the MS/MS filtering to a DDA strategy, I attempted that without success.

Any help would be appreciated. I've uploaded a minimal Skyline project that replicates this as a .zip to Uploads Page (File Name: M.Mann Bug.zip). I imagine you'll need the raw files too, so I've uploaded those as well (File Name: M.Mann Raw Files.zip).

Thanks regardless! Hopefully this can be resolved easily.

Morgan Mann
University of Wisconsin-Madison
Brasier Lab

mwmann responded:  2019-10-10

Just a small update because I can't sleep on this.

I took a look at the MS2 spectra in SeeMS, and found that the MS2 that skyline shows are represented perfectly (and exclusively) in SeeMS. Nevertheless, I still have these ID spectra in between. Is it possible that they are stored differently? Whatever is different, clearly Metamorpheus is at least able to access them, otherwise I wouldn't have gotten the IDs.

Nick Shulman responded:  2019-10-10
Thanks for sending those files.

When you specify that your acquisition method is DDA, Skyline will include an MS2 scan in the chromatogram if the isolation window of the MS2 scan matches any of the precursor m/z's that Skyline has calculated for the isotopes of that peptide.

So, for your peptide KSTGGKAPR++, the precursor has a monoisotopic m/z of 542.3115. Given the predicted isotope distribution of that peptide, Skyline will consider precursors that have these m/z values as well:
M-1 : 541.81
M: 542.31
M+1: 542.81
M+2: 543.31
M+3: 543.81
(Skyline includes the M-1 mass as well as any other mass in the isotope distribution whose abundance is at least 1%).

In your screenshot there is an ID at 33.56 minutes, but there is no point included in the MS2 chromatogram.

The reason for this is that the isolation window of that scan (Scan #9942) is 541.32. That m/z would correspond to something that was 2 Daltons ligher than the monoisotopic mass of your peptide.

Do you think that your peptide search engine misidentified that scan? I cannot think of a reason that the peptide would appear with a mass that was that far off.
-- Nick
mwmann responded:  2019-10-10
Okay, I see what you're talking about. Metamorpheus's list of PSMs lists 542.31 as the precursor mass at that scan (I've attached a copy of the list, with only PSMs matching to this peptide), so I have two guesses:

1. It has something to do with the calibration step that Metamorpheus uses (Unlikely, I think; it would have to effect a lot more spectra).

2. The Isolation window for Scan 9942 is [539.83337-542.83337], which includes the correct precursor, and may have allowed an ID, and certainly explains why I can see the right fragments.

If it is case two, is there any way to include MS2 scans which have my intended precursor in their window (as opposed to being the center of the window)? Maybe one of the DIA isolation strategies? I guess didn't try them all.
Nick Shulman responded:  2019-10-11
Skyline is not the right tool to quantify from individual MS2 scans in a DDA experiment.

Skyline insists on drawing a line between the chromatogram points, and calculating the area under that line. Because of that, your acqusition method has to tell the mass spectrometer to collect spectra with exactly the same isolation window at regular intervals, either as a PRM or a DIA method.

Peak intensities in MS2 spectra with different isolation windows cannot easily be compared to each other because the scans will isolate different portions of the precursor's isotope envelope.

If you can identify the spectra that you want Skyline to use for drawing chromatograms you might be able to get Skyline to do what you want by telling Skyline that it is a DIA method, but you might have to use something like ProteoWizard MSConvert to remove the spectra that you want Skyline to ignore.

-- Nick
mwmann responded:  2019-10-11
That makes sense. The conclusion that I'm drawing from this is that my mass preference list didn't work (or didn't work as intended), and that the extra ID times were just a happy accident. I'll have to figure out what I did wrong on the acquisition side to fix this for the future.

Thankfully, I'm likely to follow up with an MRM anyway, since there's a PTM my PI is especially interested in that was too low abundance to quantify without an isotope standard. This should give me an opportunity to fill in the blanks for any isobaric species that I couldn't resolve chromatographically.

Thanks for the help! It really cleared things up.