Absolute concentration question

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Absolute concentration question jung165  2019-01-28
 

Hello Skyline team.

I have a quick question about absolute concentration.
I followed the absolute concentration procedures with https://skyline.ms/announcements/home/support/download.view?entityId=aded0ebd-d12e-1034-84af-a631c495ef40&name=Skyline Absolute Quantification.pdf&_docid=attachment%3A%2Faded0ebd-d12e-1034-84af-a631c495ef40%2FSkyline%20Absolute%20Quantification.pdf, that Skyline provided.

So, my question is that light peptides are required for absolute concentration?? Is there any other way to measure absolute concentration with only heavy labeled SIS peptides and those endogenous samples??

I appreciate for your help.

 
 
Brian Pratt responded:  2019-01-28
This is probably what you're looking for: https://skyline.ms/wiki/home/software/Skyline/page.view?name=Surrogate%20Standards

Best Regards,

Brian Pratt
 
Nick Shulman responded:  2019-01-28
In the Absolute Quantification tutorial, the heavy labeled peptides are just used for normalization.

There are other ways of doing normalization in Skyline, and you could even choose not to do any normalization at all in Skyline if your experimental procedure is consistent enough.

You can tell Skyline which normalization method to use at:
Settings > Peptide Settings > Quantification

Skyline will calculate the calibration curve from your external standards. External standards are samples where you have spiked in a known amout of your analyte. Skyline will do a linear regression between the concentrations that you have specified for those standards, and the observed peak areas in order to figure out the conversion between peak area and absolute quantity.

Does this answer your question? If not, maybe you could post a picture of what you are looking at.
-- Nick