Thank you for sending me your files by email.
I would say that the reason that the peaks look bad is that Skyline was unable to find your peptides of interest.
Skyline always tries to choose the best looking peak, which does mean that if they peptide cannot be detected that Skyline will end up choosing a very bad looking peak.
Since your raw files only contain MS2 PRM spectra, it is difficult to say what exactly is going wrong.
The problem might be that the peptides that you are looking for really are not in the samples, or are in such low abundance that they cannot be detected by the mass spectrometer.
The problem might be that the peptides are there, but the mass spectrometer was miscalibrated so that the MS2 isolation window was in the wrong place to capture your peptide of interest for fragmentation.
If you are able to run this experiment again, there are some things that you might be able to do so that you get have more information if things go wrong:
1. Tell the mass spectrometer to collect MS1 spectra in addition to the PRM spectra that you have told it to collect.
2. Spike a known peptide into the sample and tell the mass spectrometer to collect PRM spectra for that peptide.
3. Spike a known intact protein into the sample and tell the mass spectrometer to collect PRM spectra for some of the expected peptides from that protein.
If you are able to detect the spiked-in proteins and/or peptides, then that will give you more information as to whether your problem is with sample preparation, mass spectrometer calibration, etc.
I do not have any practical experience using a mass spectrometer so my advice might be of limited value, but I hope this helps.
-- Nick |