The single cell proteomics revolution!


There are seriously 10 papers open on my desktop that I want to blog about — and will! — but I'm busy, so time for another super lazy post.
Last year some cool people asked me if I'd be interested in doing some articles about things happening in proteomics that I absolutely thought the outside world should know about. My first thought?!?? Single cell proteomics (by SCoPE-MS).
This is the best I could come up with.
(Of course, I love to type, so I also talked about the study I credit with making proteomics a reality for the rest of us.)
On this topic, I recently was so sleepy that I went through all the "comments" on my blog. There was around 2,000 spam messages suggesting all sorts of terribleness, but there were also some legit comments. And — I tell you what — SCoPE-MS gets some comments. Particularly regarding aspects of the RAW data in the public repositories, and I think that is something we will really need to talk about at some point.
My opinion is that we've been really lucky as a field in that we….mostly haven't actually been sample limited. Ten years ago the people doing cell culture would look at me like I was a tyrant when I said I needed 1mg of protein for global + PTMs. I get the same exact look now when I ask for 50 micrograms.
With the exception of PTMs on tyrosine, glycopeptides and a few other weird things, I'd feel comfortable saying that >90% of the peptide MS/MS spectra reported in the literature have looked like this —

>80% sequence coverage thanks to
1) An abundance of signal
2) Really really friendly charge distribution thanks to basic residues
In SCoPE-MS we don't have #1. There is a limit to how much you can load your carrier channel without fogging your single cell signal (as an aside, I have a crazy hypothesis that this limit is very different depending on whether you are using a D20 or D30 Orbitrap). So…the spectra are always flirting with the background noise. And…at low signal, nothing is all that pretty.
Here is the big question though:
How many fragments to you actually need for confidence in that identification?
Another question: If you were doing targeted peptide stuff with SRMs how many do you need to trust an identification? 3? With unit resolution? And a good reproducible retention time?
I think we've got a philosphical hurdle at some level for this one, particularly for people in our field with Analytical Chemistry as their background. If you look at who got really comfortable with the SCoPE-MS stuff and jumped on it first, I think it has been the people who are coming from the genomics or informatics world.
I promise, if you had been looking at microarrays yesterday, the SCoPE-MS data is a huge and beautiful upgrade. But, if you are used to loading 1ug of peptides on your Q Exactive….SCoPE-MS data is going to take some getting used to.

عن zekal3_3

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