Wednesday, September 4, 2019
6 hour gradients + HF-X + DIA = 10,000 Human Proteins
...well....maybe I'll interpret the peak width stuff a little later....BECAUSE EVERY INDIVIDUAL FILE IS OVER 15GB!!! (...well...at least the ones I'm interested in, from this study based on the results they report)
The title of this post kind of sums it up.
This team looks at several different chromatography conditions and materials to gradually build an ideal gradient for their ultra-long run DIA analysis. I think they settle on 60cm column of CSH solid phase at 250nL/min. This is probably a really good idea because they use a slEasyNLC 1200 system and at higher flow rates you'd probably run short of buffer.
0.3 x 360 (6 hour @ 60min) = 108uL? Okay, so not as bad as I'd have thought. The total pump capacity is only 140 uL on each pump. If you assume that you use around 12 uL to load (didn't look, but that's typically what I expect) you're still okay.
They use an HF-X system with 120,000 resolution MS1 and it looks like 30,000 resolution DIA windows, but 60,000 when the gradient gets to 6 hours and beyond. 60,000 resolution scans take a long time. Your peaks are gonna shift by the time you get through a full cycle. To compensate for this, they throw in an additional MS1 scan part-way through to allow AGC to have better data to work off of.
MS1 (AGC calculation) - DIA/DIA/DIA/ MS1 (AGC calculation) DIA/DIA/DIA/DIA - Repeat (number of windows not accurate)
3 normalized energies are used -- 25.5, 27, and 30. I find this surprising because a lot of the recent DIA work I've seen has used direct eV for the fragmentation since the normalization doesn't do much. This is easier, and it's interesting to me that such a small step is worth the effort of putting it in!
SpectroNaut is the data analysis software and they do some interesting stuff with the data processing. In some experiments they rely on a library made directly from .FASTA, though it looks like ultimately the best data is obtained when they use it in combination with real libraries.
I'd hoped to look at the RAW data, but it looks like my ConCast home internet has said no. I've got 4GB downloaded and it still says 2 hours for one file. If you're interested in DIA there is a solid amount to learn from this new study.
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