Tuesday, November 1, 2016

TKO TMT Standard for assessing isolation interference!

Isobaric tagging things like TMT and iTRAQ suffer from "isolation interference" it is a consequence of the technology. You can mitigate it by improving the resolution of your chromatography, with apex peak selection and by using MS3 based techniques.

Post processing software can give you a decent metric of your co-isolation interference, like this screenshot from PD 2.1...

(here this is a TMT10plex 2D separation I did a while back. This metric takes the total signal intensity of your selected ion divided by the total signal intensity of the MS1 window)  and this is a useful metric.

Joao A. Paulo et al., suggest an alternative methodology.   They describe how to create a standard by using deletion strains you can purchase from the ATCC that are described in the Yeast Deletion Collection.

The strains they choose have proteins that are completely absent, so if everything was perfect you'd get zero signal from all the peptides from these missing proteins in the appropriate channel!

I could see this being used a couple ways:
1) To optimize your parameters till you had parameters as close to zero signal as you could possibly get
2) To use as a pre- or post- run control to have the best understanding of how much you can trust your actual experiments!

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