Hypothetical multiplex tag works in single cells?

 

Did you know there are other tags out there for multiplexing proteomics? Younger people probably don't and I can't tell you where they to get them because I actually truly can't. Let's change the subject entirely.

Did you know lawyers are seriously expensive? Like, for real expensive. If you're struggling with science salaries maybe you should check it out. Okay, let's go back to this paper.

If hypothetical multiplex tags did exist in some places where I couldn't tell you about could those ficitious tags be used for single cell proteomics?  Are you thinking....ummm...yes...? why wouldn't they be? These people found a team of peer reviewers who thought it was useful to check - at Analytical Chemistry! 

And they compared it directly to the commercial reagents that we all know and love. They used the same intrument Orbitrap Fusion Eclipse using 120kDa MS1 and 30kDa MS/MS. The only difference is that the fictitious tags that don't exist and if they did I couldn't tell you where to get them used a slightly lower m/z cutoff. They also optimized at 5:1 tag to peptide. 

For the experimental design here, they sorta mailed it in. 3 tags were used for cells and other tags were used for different controls. They also optimized the carrier to single cells by basically not saying "...why would it be different for one multiplexed reagent when 15 different papers already optimized this on the same Orbitrap hardware...? and said you could go about 100x - 200x to one? 

Then they actually did some interesting stuff by labeling mouse spleen cells with 13 of their available 16 channels. The most interesting part is where they find that if they don't use FDR at all ("set to 100%") they can get 12,543 proteins in mouse spleen cells!!! Someone said "ummm....wtf....you need to use FDR..." and they get 3,991 peptides and 3,602 proteins. So....1.1 peptides/protein on average. Ouch. The FDR calculation scheme is ...nonconventional.... and I almost want to download their data and reprocess it in FragPipe and see if the data is good but the data analysis is unnecessarily strange. Oh. The fun I had when I had free time....

Interestingly, however, the authors get those 3,602 one-hit-wonder proteins to clearly separate the different cells in the mouse spleen into their originating cells, and generate a beautiful T-SNE or U-Map plot, and that's what we came here for anyway, right? The authors suggest some follow-up experiments where they plan to combine both their tagging solution with the amazing commercial one....