Wednesday, March 22, 2017
MvM workflow -- Combine DDA with DIA!
This one takes a second to wrap your brain around -- but...to get to proteins that are only estimated to be expressed at 50 copies/cell(!!) it is worth it.
The paper is brand new and can be found at JPR here.
The basic idea is that if you run your normal DDA (TopN-type) experiment, you can break the peptides coming off the column into 3 groups:
Group 1 -- Fragmented and identified in all runs and any label free algorithm will give you amazing quantification
Group 2 -- Fragmented in a few, but not all runs. Identified, but you'd have to infer (or impute) their identity from MS1 only in the other runs
Group 3 -- Peptides you never fragment that are just too low in abundance to ever crack the N-most intense in your TopN experiment
The MvM strategy (Missing Value Monitoring) specifically focuses on Group 2. You have this subgroup of peptides that have been identified -- which means you have a Peptide Spectral Match (PSM) that you can use to create a spectral library.
If you then run DIA on every file you can use the spectral libraries you made to quantify the peptides with missing values across all of your runs.
To test this strategy, this group uses a QE (the paper says QE HF, but the method section uses resolutions that show it as a QE Classic or Plus) on a yeast cells during different stages in their developmental cycle or something. They are able to get incredible depth, with even lowest abundant proteins being quantified in all samples.
Up-side -- This approach doesn't use any funky software and you get much better label free quan!
Down-side -- You need to run every sample for both DDA and DIA.
I really like this paper because it is a clever approach I haven't considered before. If the queue in your freezer seems to be growing at an ever faster rate, this might not be the LFQ method of your dreams ;)
But...if you have the available instrument time that you could run each sample twice, this might be a great method to consider!