|peptide absolute quantification from PRM data sanity check||manguy jean||2022-09-23 07:44|
I learned how to use Skyline on the job for a peptide absolute quantification project. I read a lot of the tutorials, some of the posts I found here from different google searches, and looked at the methods sections of diverse papers. I think what I did makes sense I just want to make sure I didn't make a mistake because of some blindspots, so any help or advice is welcome.
We want to quantify some peptides from a hydrolysate (not for protein quantification but just for peptide quantification so we didn't have the choice of which peptides would be best). We have heavy variants of each of them. We built an internal calibration curve for each peptide using varying concentrations of heavy peptides. The first series of runs (PRM on a QE) uses a wide scale of concentrations to find the approximate concentration of light peptide, then another series of runs to adjust the calibration curve for each peptide to quantify and then replicated that again.
I used Skyline to analyze these data. I used the retention time of these peptides in previous DDA runs using the same LC parameters and the presence of peaks for the heavy and light products to select the correct peaks. Removed the product ions interfering with the peak. I then extracted the ratio of heavy/light for each analytical replicate and for each concentration. I used these ratios to make a calibration curve (linear regression) and I looked for the ratio heavy / light == 1 to find out the concentration of light. I had to go outside Skyline for this step as all my samples are marked as
I hope someone can tell me if I am completely off the tracks or not.