Sunday, January 7, 2018
DIA of clinical serum samples!
There are a number of aspects of this new study that I like!
The team recognizes up front how critical minimization of sample handling is going to be as we move more proteomics technologies into the clinic. In this work they use a version of SISPROT (the original method is described here and is worth taking a look at!). The closer we can get to going straight from a biological material to peptides without putting a pipette tip in it the better, right?
Pooled samples were depleted and fractionated to develop a comprehensive spectral library for the study. I'm still on the fence about serum/plasma depletion and have tried to keep up with what everyone else is doing over the years (some old blog posts on the topic are here, here and here) and I hope to do some comparisons on modern instruments myself soon, but I really like the fact that it was used for spectral library generation, but was not used on the actual patient samples.
The SISPROT eluted peptides were combined with the iRT internal standards from Biognosys and a number of DIA variations (different windows and gradient lengths) were tested. The team lands on a 50 minute nanoLC gradient being ideal and shows that their results compare favorably to one of my favorite recent DDA studies (the Max Planck clinical weight loss one that is here). I had to go to ProteomeXchange and get a RAW file. For some reason I thought the method was 20 minutes on the QE HF. But the two are very similar in terms of total run time. 44 min vs 50 min.
50 minutes isn't bad. But it's still going to be a tough sell for most clinics where they've got to justify sample throughput. We're getting there, though!
How did this DIA group do? Pretty great! They quantified about 400 proteins, including 50 of the FDA defined biomarkers. They get 5 orders of magnitude in quantification which is more than I would have guessed. And they did all this with a Q Exactive Classic. This is a big deal because as nice as the Max Planck study is, the HF is going to be a tough purchase on a hospital budget. Now that the Classic can be picked up for the same price or less than many triple quad instruments, it's less likely to sticker shock an administrator into shutting the whole thing down.
I gotta try out the new SpectroNaut soon....