Global vs Surrogate standards

support
Global vs Surrogate standards sstoychev  2017-12-19 00:30
 
Dear Skyline team, could you provide some information on the differences between Global/ Surrogate/ QC standards when it comes to sample normalization. How do these work i.e. are the peak areas of peptides set as Global standards averaged or summed prior normalization.

Also, please have a look at the attached screenshot. Why is the "standard selection" window grayed out hence not allowing for selection?

Regards,

Stoyan
 
 
Nick Shulman responded:  2017-12-19 01:16
In your screenshot, the reason that you cannot change the standard type of that particular peptide is that the peptide is one of the iRT peptides in your iRT calculator. (You can tell this because of the clock overlay on the peptide icon). You can change whether that peptide is an iRT peptide by going to
Settings > Peptide Settings > Prediction

"QC Standards" have nothing to do with normalization-- if you have a multiple injection replicate (that is a replicate with more than one .raw file in it), then, when you mark a peptide as a QC standard, Skyline will expect to be able to find that peptide in every one of the .raw files in the replicate.

When Skyline shows you a calibration curve for a peptide, Skyline needs to know how you want that peptide normalized. The default normalization method for your Skyline document is controlled by the Normalization Method setting in:
Settings > Peptide Settings > Quantification

You might need some of your peptides to be normalized differently than the other peptides. In the Document Grid, there is a "Normalization Method" column available on Peptides which lets you override the Normalization Method for individual peptides.

In the small molecule world, sometimes you want to normalize a particular analyte molecule against a different standard molecule that is not expected to have the same retention time as the analyte. In order to achieve this, you would mark the standard molecule as a "Surrogate Standard", and, then, "Ratio to Surrogate xxx" would become available in the Normalization Method dropdown on the Document Grid. (There's an overly concise description of Surrogate Standards here: https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards )

You can also specify certain peptides as "Global Standards" whose peak areas get summed and are used for normalization when the Normalization Method is "Ratio to Global Standards".