Wednesday, September 26, 2018
Splitting up TMT kits? Skip every other channel and increase your coverage!
We don't always have 11 samples to run. In cost per reaction though, (at least with my old sales rep -- the new one doesn't seem to appreciate the level of discount I expect 💔😇😇) TMT-11plex is about the cheapest way to go.
If you don't have 11 channels, for example, you have 6 -- you can get a boost in your number of peptide and protein IDs by skipping every other channel.
If I have 6 samples I will pick an N or C variant for each unit mass and stick to it. For example
And make sure to not mix in any of the C variants from the same unit mass. To fully resolve 129N/C you need 43,225 resolution @ m/z of 200. This means you need to use 50,000 resolution on a Tribrid, QE HF or QE HF-X. Or 70,000 resolution on a QE or QE Plus.
Sooooooo slooooow..... Great data...but...sloooooowww....
If you skip the N/C variants for each unit then your reagent is essentially the TMT6-kit (with shuffling of the N/C)
Worth noting -- if you are using Proteome Discoverer you probably want to create a new quan method for the channels you use -- if you want PD to normalize the data
-- if you aren't normalizing and/or imputing quan you probably don't need to worry about it. You can just hide those channels in the output report. What you don't want is background noise in your quan region (or a reporter M+1 isotope) being mistaken for your quan, it being scaled up as if you loaded only 1% of peptide in channel 128C and some noise being amplified 100x. Probably harmless in the end, but it totally looks weird and worries me that it might actually affect something.