I do like it when a new group gets on the the single cell proteomics train and starts optimizing/reoptimizing things. Despite the 300 reviews, 50 method optimization papers and 20 biological studies that have been published, each new one brings a new perspectice and observations.
While I don't love every aspect of this paper (some insight on what LC gradients were used when seems to be entirely absent from the main manuscript, which makes me question the title which seems to describe a single workflow) there is some gold in here!
In my lab we don't reduce and alkylate the single cell derived proteins. I do this because I'm lazy. And also because I spend a lot of time studying drugs that modify cysteines.
A really nice evaluation of different reduction and alkylation conditions in this study finds optimal conditions and reagents for reducing and alkylating. However, the %CV decreases when doing so under most conditions, probably due to the extra manipulation steps. This section is really well done.
This group also looks at how to digest single cell derived proteins at 37C without those teeny tiny droplets of trypsin evaluating and comes up with a method that works well. They also describe an easy way to get to 100% humidity.
Since they're using a nanoelute they can go between 96 and 384 well plates. Simply moving from the 96 - 384 well plates is enough to give them 300 protein groups!
Multiple cell lines were used here. A549 cancer cells, primary astrocytes grown on poly-lysine, etc., it and the system in use is a TIMSTOF SCP and data was processed in DIA-NN 1.8.2 (?) Even if you ran your first single cells (eek. 8...or 9...? years? ago?) There is probably something to learn or re-learn here. I'm certaily adding it to my lab's Slack channel for literature now!
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