I want to calculate the area under the curve of the extracted ion chromatograms of my mass spectrometry dataset using Proteome Discoverer (Thermo Fisher). However, the area shows as zero for all identified peptides. Individual XICs look good and the workflow is exactly as instructed. Attached screenshots show my workflow, and other results.
Does anyone with knowledge of Proteome Discoverer have any clue where things might have gone wrong?
Alexander,
I don't have a direct answer to your question, but I can confirm that we found this software sometimes to be buggy, too. If the support team cannot help you, you may consider to use Skyline software from Mike MacCoss' lab to calculate your XICs. We have better experiences with that.
Sonja
You can have a look on thermo omits portal as we (and others) had a similar problem when using PD 1.4 (https://portal.thermo-brims.com/index.php?option=com_discussions&view=thread&catid=4%3Aproteomics-forum&thread=2252%3Amissing-precursor-ion-area-detector-values&Itemid=54 and http://portal.thermo-brims.com/index.php?option=com_discussions&view=thread&catid=4%3Aproteomics-forum&thread=2277%3Aprecursor-ion-area-detector-area-calcualtion-problem&Itemid=54). We had only some peptides with missing values. Also, from what I know, PD 1.4 can display a precursor area information for a protein (with Precursor ions area detector node) only from the average of top 3 most abundant peptides, even if those peptides are not unique and until now, I did not find any option to modify this. You could also use MaxQuant, a program that can report the intensity, LFQ and iBAQ value for a protein and you can choose what peptides to consider for quan (unique, razor, modified, unmodified etc.) or Skyline as Sonja suggested.
Regards,
Cristian
Thank you, Sonja and Cristian for your answers. It appears Service Pack 1 of PD1.4 partly solves this issue. It generates peak areas for most of the peptides now.
However, some peptides still have no peak areas for some reason while multiple PSMs of other peptides show different areas, where they should be the same. I have taken this issue to the Thermo omics portals now.
I could be wrong, but I believed MaxQuant was only suitable for labelled quantification and we do not use labelling in our experiments. Also, I have not been able to do this in Skyline could anyone point me to a guide or tutorial on how to do that?
Thanks for the help!
Dear Alexander,
I would be interested in solving the problem of missing precursor area for some peptides in PD 1.4 as I have no definite answer for this (https://portal.thermo-brims.com/index.php?option=com_discussions&view=thread&catid=4&thread=1972&Itemid=54).
Regarding MaxQuant, the program was initially developed for SILAC based experiments, but at this moment does have support also for label-free experiments (http://www.maxquant.org/faq.htm) and a paper with label-free (http://www.cell.com/immunity/abstract/S1074-7613(10)00044-0), but there also others.
I do not have any experience in using Skyline so perhaps Sonja can help you with a tutorial.
Regards,
Cristian
Hi Christian,
I have attached 3 screenshots of typical problems. You can see a lot of missing areas in the screenshots.
The first screenshot shows a missing area with a generally bad XIC. I have quite a few of them, to be honest, and I do not realy know if XICs like these are common or not. Are these peptide id's trustworthy or are these typical false positives?
Second screenshot shows a good XIC with still no calculated area. Usually, XICs like this give good areas, but some still don't.
Last screenshot shows a peptide (KVLAIDHLNEDQLR) which is probably "sticky" as it has multiple samplings. There are a lot of instances (mainly in the large shoulder) that causes it to not calculate an area, but there are also a lot of calculated areas which differ from each other.
Do you recognize these problems? I read your topic on thermo-brims. Did you get any useful information from the thermo staff?
Last question: do you work with or without peptide grouping? How does PD calculate the peptide "area" from all relevant PSMs when peptide grouping is on?
Alexander
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics
- Bioinformatic Tools