short question absolute peptide/protein quantification with internal heavy labeled Tobi  2019-01-03 05:09
 

Dear all, wish you a good start into to the new year.

Is there a way to average peptide quantification results directly within skyline into protein quantification results? I can imagine this beeing troublesome in cases where peptides belong to several proteins, but when working with preselected representative peptides this might be helpful to quickly get to a complete results report. Do you have plans to implement something in this direction or do you want to stay on the peptide level?

Question on internal standard based absolute quantification.

Is it possible to do internal absolute quantification with different amounts in each replicate/file? So far I only get no or messed up quantitative results based on some regressions which I might not be able to avoid if I want the quantification result at all.

I would like to set to Replicate/Analyte Concentration to values ranging from 115-140 fmol, meaning in replicate 01_1 all heavy labeled peptides are present at an amount of 115fmol, while in replicate 04_01 the value is 140 fmol etc. All light peptides should be quantified against heavy but only within the same file.

As far as I can see I don't get the expected output for Quantification in Peptide Ratio Results. All light BSA peptides should have around 100 fmol for all replicates, while spectrin peptides should have ca. 20 fmol in replicate 02_01 and 02_02 but close to 0 fmol in other replicates.

I know this might not be the usual approach but it is for comparisons and it would be nice to know if it is technically possible or not.

Best regards,
tobi
 
 
Nick Shulman responded:  2019-01-03 14:31
You can bring up the "Pivot Editor" in the Document Grid in order to calculate the average peptide area for each protein. I've attached a PowerPoint showing how to do that.
There is a little more about the pivot editor on this page:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=PivotEditor

Skyline does support calculating concentrations just from the ratio to internal standard. In the Document Grid, there is a column on the "Peptide" called "Internal Standard Concentration" where you can specify at what concentration you spiked in your heavy form of that peptide. In the Document Grid, there's a view called "Peptide Quantification" which includes the Internal Standard Concentration.

In order to use this Internal Standard Concentration to calculate the absolute quantification, you need to tell Skyline not to try to create a calibration curvce.
So, on:
Settings > Peptide Settings > Quantification
you need to set "Regression fit" to "None".
You also need to set "Normalization Method" to "Ratio to Heavy".

You can tell Skyline that you different replicates have been diluted by different amounts using the "Sample Dilution Factor" column (it's on a Replicate). The purpose of this column is for handling the case where some of your replicates were diluted by different amounts in order to bring the measured intensity into the linear range, but it also works in the case where you want an extra number to get multiplied in before arriving at the Calculated Concentration value.
I am not sure whether the "Sample Dilution Factor" should be set to those concentration (115, 140, etc) or should be their reciprocal (0.008695652, 0.007142857, etc). You can try it both ways and see which one results in the correct values for the Calculated Concentration.

The Calculated Concentration column is available in the Document Grid. It's under:
Proteins > Peptides > Peptide Results > Quantification > Calculated Concentration
-- Nick
 
Tobi responded:  2019-01-06 08:59
Dear Nick,

thank you very much for the templates and the guidance, it work out nicely.

It was just that by defaul the column Replicate/ Analyte Concentration seemed to be the required element but it did not work in my case at all. In the pop-up explanation for this, maybe add the hint of using Peptides/Internal Standard Concentration plus Replicate/Sample Dilution Factor?

To sum up the fix:
Setting a fixed value for internal standard under Peptides/ Internal Standard Concentration and a reciprocal factor under Replicates/ Sample Dilution Factor works nicely also for the Peptide/ Calculated Concentration averaged into protein calculated concentrations with the pivot template.

Thank you a lot