Question New Protein Abundance Report Feature

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Question New Protein Abundance Report Feature roman sakson  2020-10-12
 

Dear Skyline team,

thank you for providing us with the possibility to get numbers representing protein abundance per replicate directly from Skyline, which is also supposed to be part of the next main Skyline release. I intend to use this option rather frequently in my research and would like to understand it a bit better. I am mainly interested in the normalization case against heavy (each light signal must have a heavy counterpart to be considered).

In the release notes, you state that this protein abundance value is the same that the Group Comparison (GC) framework uses. I thought that, as default, on protein level GC sums up all transition areas that belong to the same protein (also across different peptides) that are quantitative and have a heavy counterpart and then normalizes this sum to the sum of heavy signals. This is slightly different compared with calculating normalized ratios transition by transition and then averaging those ratios, since strong signals dominate the sums of values, as previously described on several occasions. However, the explanation in the reporting window for the "Protein Abundance" feature now states that "The Protein Abundance is calculated by taking the average of the normalized areas of all of the Transitions under the Protein." This now sounds to me exactly as the latter option to do it and the question is, whether protein abundance numbers really are exactly what the GC framework uses?

Thanks a lot,

Roman

 
 
Nick Shulman responded:  2020-10-12
Yes, the protein abundance number is exactly what the group comparison uses.
If you are doing ratio to heavy, then that number is calculated by taking the sum of the light transition areas across all of the peptides and dividing by the sum of the heavy transition areas.
However, transitions are only included in these sums if the matching transition is present and has a valid peak area on the other side of the dividing line.

-- Nick
 
roman sakson responded:  2020-10-12
Hi Nick,

ok, thank you! I guess I got a bit confused by the "taking the average of the normalized areas of all of the Transitions" statement in the report help but I am happy to read that it is still the sum.

Unfortunately, I also have a practical question: I created a document grid report to extract the protein abundance values in a document that contains only one protein, consisting of one peptide with a heavy and a light precursor present. I have 6 runs grouped into two conditions. For GC, I can normalize to heavy or not and everything works as usual. However, if I go to Peptide Settings and set "Ratio to Heavy" as the normalization for quantification, all my protein abundance values become #N/A. For no normalization or equalize to median, everything works and I get reasonable values. Do you have an idea what might be wrong and how I can get abundances normalized to heavy? I have also observed this in other Skyline documents (I am using the latest daily release). The document is shared below.

Roman
 
Nick Shulman responded:  2020-10-12
Oops. I wrote the "if" statement backwards. The behavior was supposed to be the opposite: The protein abundance should be #N/A if there are missing transitions, _unless_ the normalization method is Ratio to Heavy.

I will fix this. This fix will probably appear in the first Skyline-Daily which comes out after we release Skyline 20.2, which will probably be later this week.

Thank you for reporting this bug.
-- Nick
 
roman sakson responded:  2020-10-12
Sure, I am happy to help!

Thank you,

Roman