peptide count from document grid plus pivot not counted correctly

peptide count from document grid plus pivot not counted correctly sas28  2019-04-18

Hi skyline team,
I am exporting total sum area for each protein using your document grid with the pivot function. As an addition I want to know how many peptides per protein were used for the summed area. I have noticed that there are several options for total area in the drop down menu and I went for Proteins -> Peptides -> Precursors -> Precursor results -> total area which gave the same result as protein -> peptides -> peptide results -> quantification -> normalized area. However when adding peptides as count in the pivot function, it doesn't accurately reflect the peptides that are actually in the Skyline document. For example for iRT peptides which i have got 10 of, it reports 18 in the final exported summed area document. Other examples are only 1 off and sometimes the peptide count is correct. What am i doing wrong? Many thanks.

Nick Shulman responded:  2019-04-18
When you include the "Total Area" column on your report, you end up getting one row per Precursor Result.
The "Count" operation in the Pivot Editor tells you the number of rows that did not have a blank value in that column. It does not do anything smart, such as figuring out how many of those values are identical to each other.
For this reason, that "Count of Peptide" column ends up being the number of precursors under the protein, which is slightly higher than the number of peptides since some of your peptides have more than one charge state.

I think if you really started with a report that only had the following columns:
Normalized Area
and did the pivoting, you would end up with "Count of Peptide" being the correct number. But, if that starting report has any precursor columns (e.g. "Total Area") it will end up throwing off the final count.

If you wanted to use "Total Area", you could still do it. In this case, you would start with a report with the following columns:
Total Area
and then pivot it, with "Protein", "Peptide", and "Replicate" as Row Headers, and Sum of Total Area as the value.
Then, you would pivot it again, with "Protein" and "Replicate" as Row Headers, and "Count of Peptide" and "Sum of Sum Of Total Area" as values.

-- Nick
sas28 responded:  2019-04-19
Great. Many thanks for such a speedy reply.