Is L/H ratio corrected for shift in isotope patterns?

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Is L/H ratio corrected for shift in isotope patterns? Tobi  2018-12-05
 

Dear skyline team,

reading this nice technical note ( http://www.isotope.com/userfiles/files/assetLibrary/PRT_MACCOSS.pdf ) brings me to ask if and which corrections skyline applies to SILAC ratios when quantification is not based on the whole isotope cluster but just on the precursor or fragments from isolating just the precursor. (want to make sure i dont accidentially apply correction twice)

The issue is well illustrated in figure 3 of the linked pdf, stating that the shape of the heavy peptide is shifted towards the precursor with the heavy precursor intensity beeing up to 10% higher in a 1:1 light:heavy mix (depending on the type of labeling). Adding up intensities of precursor plus isotopes before calculating the ratio evens out this shift, but this is not happening fully in PRM and quantification on the precursor level.

It would be great getting a short reply.

Best, tobi

 
 
Nick Shulman responded:  2018-12-05
Skyline does not do any sort of correction for this when calculating light to heavy peak area ratios. It looks like figure 3 is talking about the difference in the shape of the isotope distribution caused by the fact that the heavy amino acids were only 99% labeled. The other sort of error that Skyline does not correct for is if the two isotope distributions overlap (for instance, when the heavy amino acid is only 3 Daltons heavier than the light one).

We have plans in the future to do this sort of isotope distribution aware correction, but we do not do any now.

Skyline does allow you to specify the Atom Percent Enrichment (APE) for the heavy labels, but the only place that that actually makes a difference is when Skyline is calculating the isotope dot product. You specify the APE at:
Settings > Transition Settings > Full Scan > Isotope Labeling Enrichment

-- Nick
 
Tobi responded:  2018-12-05
Dear Nick,

thanks for the response on the isotope product and the 3 Da shift. Just for figure 3 i think it represents something completely different, since labeling efficiency is indicated at 99.9%, and a poor labeling efficiency would not result in a higher heavy precursor peak.

Its more that a molecule with e.g. 60 carbons has a defined isotope pattern, but when e.g. 10 carbons are fully labeled (Arg10) the isotope pattern of the heavy molecule should look like from 50 carbons instad of 60, which leads to a higher P/M+1 ratio for the heavy labeled. I did some estimations in excel and if a labeling of 100% is assumed, an Arg10 SILAC pair of 1:1 molar ratio should have an 11% higher precursor area (similar as in the technical note and the 11% should be constant for alll molecules with Arg10 label).

Sounds quite high to me, looking forward to see what will come in the future.

Thank you very much.

Best, tobi