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Absolute quantification calibration curve

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Absolute quantification calibration curve mvm35  2024-12-19 03:58
 

Dear Skyline,

I am trying to generate a calibration curve using heavy and light peptides in a background matrix that contains my endogenous light peptide.

Could you provide guidance on what I need to do.

Conventionally I would spike in a constant heavy amount of peptide with changing light peptide in a constant background matrix conc. However, my matrix contains endogenous light peptide.

What would you suggest? Keep light peptide spike in constant and vary the heavy peptide?

Many thanks,
Mehul
 
 
mvm35 responded:  2024-12-19 04:09

Do I need to even spike in light peptide if my matrix already contains a constant amount of light peptide?
 
Nick Shulman responded:  2024-12-19 07:45
Here is another support request where someone asked about reverse calibration curves:
https://skyline.ms/announcements/home/support/thread.view?rowId=66481
-- Nick
 
mvm35 responded:  2024-12-19 09:08
Dear Nick,

thank you for your help could you checking my thinking bellow and confirm.

I will make a reverse calibration curve without spiking in light peptide and use the matrix endogenous light peptide for normalisation against changing heavy spiked in peptide.

The above will be used to assess the range in which there is a linear response from the peptide and as such a single point spike in with the linear range will be used for quantitation.

Many thanks,
Mehul Makwana.