Absolute quantification with oxidized peptides

Absolute quantification with oxidized peptides clemence balty  2023-08-25 04:39

I'm doing absolute quantification using the SureQuant method. I have a peptide with a methionine. I wonder if it would be possible to calculate the quantity of this peptide based on the sum of the reduced peptide and oxidized peptide. I mean set a calibration curve with the sum of both area in fonction of the quantity. Is there a way to do this in skyline or should I do it manually ?

Nick Shulman responded:  2023-08-25 05:20
There is no way to do this in Skyline right now, but I am supposed to implement this feature soon.
Specifically, the feature that I am supposed to implement is "Protein-level calibration curves" where Skyline calculates the calibration curve for a group of peptides.
In the Document Grid, there is a column that you can add to your report called "Protein Abundance". That column combines the information for all of the peptides into a single value. That is the value that we are planning on having Skyline use in the future Protein-level calibration curve feature, but for now, you would have to do that manually.
-- Nick
clemence balty responded:  2023-08-29 03:16
Thank you for your response.
I tried to add "Protein Abundance" to my report. This gave me very strange results.
For my absolute quantification I have followed the absolute quantification tutorial. I have a standard curve of light (set as "standard" in the replicate document grid). Then I have an unknown replicate. In peptide settings I set Normalization as ratio to heavy and regression fit as "quadratic".
In the peptide ratio result of the document grid I have a value for my unknown replicate. When I add Protein abundance I have another result I am not confident in.
I attached the skyline file. Do you have any idea why ?
Thank you,
Nick Shulman responded:  2023-08-29 09:52
I am not sure I understand what you think is going wrong.
Can you post a screenshot of what you are seeing?
-- Nick
clemence balty responded:  2023-08-30 00:30
Hi Nick,
Please find attached a ppt with screenshots.

Thank you,
clemence balty responded:  2023-08-30 00:31
the ppt !
Nick Shulman responded:  2023-08-30 08:08
Thank you for sending the PowerPoint file.

The "Quantification" column in the Document Grid only gives you information about a single peptide. The Protein Abundance column gives you a number for a single protein. You would not expect to see the same values in those two columns.
-- Nick