Sunday, April 23, 2017
Maybe I need to give these new DIA methods a shot...
Wow....let's start there.....7,800 protein groups in 1 thoroughly optimized LC-MS run!
If I'm going to get tight CVs and MORE identifications, maybe it really is time to try getting some DIA runs queued up somewhere.
I gotta run, but you can check it out in this new open paper in Press at MCP here.
Nuts and bolts stuff? They use a QE HF, 4 hours nanoLC runs and SpectroNaut for data processing. The authors walk you through each and every step where they evaluate tweaking different parameters to find the best possible compromise between points/peak and maximum identifications.
I'm a little fuzzy on one of the optimization steps that they ended up using. The mass range for the MS1 previous to the DIA runs was splt and it led to a boost in IDs. This is a preprint paper and I think there might be a missing supplemental Excel file describing the full instrument methods. I suspect they used a rough gasphase fractionation techniuqe (half of MS1 range -- subsequent DIAs followed by higher half of MS1 range -- subsequent DIA windows.)
Stellar paper on QE HF DIA based proteomics that should make it easier for anyone thinking of doing this experiment (since they did all the optimization for you!)