What a great month for method names already! Introducing the...
...sequential single pot digestion and then sequential enrichment of the phospho- and glyco- proteome!
I'm not entirely sure what all the advantages are of the Muppet method. The authors make it seem very streamlined, and I'm guessing that you can get away with less sample and sample loss by keeping things in the tubes, but early in they have to spend a lot of time diluting urea down to functional levels. If you want a lot of the details on how this is performed you'll need to go to page 28 in the Supplemental Info PDF. There you will find that an Orbitrap 480 was used for all analysis with DIA for the peptides and phosphopeptides and DDA for the glycopeptides. So it is still 3 different injections per sample. I am always happy to see something like this, in any paper even if it's on Supplemental page 32.
I also find this a little concerning
...in Jonathan Pevsner's book (which you can get on Ebay for $12 in first or second edition), he warns that smiling volcano plots can be either a lack of data points, excessive presence/absense, or over-normalization. Since I think they've got a solid pile of data here, it does make me concerned that the data has bene over-normalized. Though...they used Bionic and specify a rather small n-glycopeptide library was used, so it could be the other two. Smiling plots just make me nervous. When I have one I generally find out I did something silly upstream.
Otherwise this seems like an interesting method, particularly if you're not always doing phosphoproteomics or glycoproteomics and you have to do them. I don't see any reason why you couldn't digest the peptides with a more traditional approach and then put those peptides into this workflow around step 2 or so.
No comments:
Post a Comment