Dear all,
I'm starting out with mass spectrometetry, and am thinking of using DIA for improved quantitation of low abundance proteins, and to save on buying TMT reagents.
Does anyone know how the batch-to-batch variation in DIA compares to using TMT and DDA?
I'll be using cells from human cell culture, and an Orbitrap Fusion Lumos.
Thanks!
Sam
Hi Sam,
your approach depends on how many samples? If it can fit into a single TMT run, and I’m pretty sure they are up to 29-plex now, then you’ll be hard pressed to beat variability with DIA. If you are running >60 samples (so now you have at least 3 x 29-plex runs) then I would argue DIA would be preferable. You clearly have the MS to do either approach. Also consider the expertise in the lab for both running the samples and analysing the data.
If you do a search, you’ll find plenty of papers comparing DIA, to TMT, to DDA. This tends not to be batch variability comparison, more coverage and missing values. There has also been a lot of work recently on removing batch affects and unless you have a shocker, it can be removed in most cases.
Good luck,
Peter
Thank you Peter G Hains ,
Unfortunately DIA is new to the lab, so it'll be a lot of optimisation and trouble shooting I expect!
I agree that most papers comparing the techniques focus on coverage.
I will likely be using around 10 samples, so TMT definitely sounds like the more straight-forward way to go!
Sam
For 10 samples - or as Peter put it, whatever you can get specifically into a single run-of-the-mill 10plex or 16plex - I would definitely go TMT, especially if you are familiar with it. Some prefractionation, and off you go.
TMT starts becoming a pain (and cost) for higher number of samples that would require merging multiple TMT batches; or if you have very different types and qualities of input. Here DIA is super-straightforward. Also you should find a good number of published DIA setups already - I would start looking e.g. in Biognosys' Lukas Reiters co-authorship publications. And the beauty is - DIA methods are fully generic as long as you stick with tryptic peptides. You can customized for individual sample types, but do not have to.
So ultimately I would say, do whatever you are more familiar with. I get more out of most samples with DIA as it is, but that is just me doing DIA since 2014.
Thank you Christof Lenz ,
At this point I'm not familiar with either technique, but I have some control cell lines that I plan to run some DIA tests on. I have a colleague who uses TMT, so that may indeed be the more straight forward way to go.
Sam
Hi Sam,
If you are specifically asking the batch-to-batch variability, then, TMT has the least amount of variability for your proteomics analysis. This is because you are running 10-11 TMT labeled samples together, as one mass spec run. As suggested above, fractionation, eg., HILIC peptide fractionation, or high pH peptide fractionation followed by LC-MS/MS will be your best approach to obtain proteomics that can access deep in the proteome, with least amount of variability between runs.
So, if you have 10-11 samples and run them separately by DIA mass spec acquisition, your missing values will be less than if you were to use DDA mass spec acquitision.. However, you will not be able to dig deep in the proteome without fractionating each sample.
So, at the end of the day, TMT will save time since you can fractionate your TMT plex once.
It is a difficult question to answer. I do not want to think about doing more than 10-11 samples since I am not convinced that you can "stich" many TMT plexes easily.
Good luck,
Hediye.
Thanks Hediye Erdjument-Bromage ,
You definitely have a point with fractionation of a pooled sample being a real strength of TMT.
I have a fellow student who takes this approach.
In terms of "stitching" TMT plexes together, she's used one of the TMT channels for a pooled sample which she runs with every batch as a reference.
Sam
- Badges
- Science method
- Similar topics
- Methodology
- Crystallography
- X-ray Diffraction