Friday, December 24, 2021

Comparing 4 phospho-enrichment strategies -- and a rant about whether we always need them!


Before I type about this great new paper comparing 4 different phosphopeptide strategies, I'd like to ramble about the fact that I've done maybe 4-6 phosphoproteomics studies in the last year and I think I've chemically enriched twice, 

Of course, you can clearly get more phosphopeptides by chemically enriching up front, but I've largely found it to be a waste of both my and my collaborator's time. (My opinion may change completely here if we're talking about tyrosine phosphopeptides, in which case I'll want to break out an antibody for pY enrichment -- or studies where the peptides of interest are super low abundance).  The best I can tell, basically only mass spectrometrists are impressed with lists of 15,000 phosphopeptides since no tools exist to perform automated analysis of crappy semi-quantitative piles of phospho sites (please correct me if this has been remedied by someone.)  If someone is fishing for phosphopeptides, I may just run a lower flowrate on my TIMSTOF using the same gradient. My S/N goes up and I process my data while looking for the phosphorylation sites appropriate for their organism. If they have a hypothesis, I use the PhosphoPedia to build PRMs for their phosphorylation sites of interest -- and we're off to the Q Exactive to do some targeting.  This isn't just because I'm not very good at sample prep. In a recent study for a collaborator with 3 x 3 biological replicates, I quantified around 5,000 proteins from her mouse organ and sent her a report with ~2,500 quantified phosphopeptides. 2hr gradient/IonOpticks Aurora/200nL/min. This isn't TIMSTOF specific, we quantified several thousand phosphopeptides from tumors from fractionated Fusion Lumos data and showed they correlated with extensively enriched phosphopeptides from the same samples. 

An increasing body of work is showing that phosphopeptides are in your global data (paper 1, paper 2)  you just have to look for them, and maybe read your audience. Is your collaborator looking for the biggest list of things possible, or is your collaborator someone who is looking for an explanation for some biological thing they're seeing. If they are the latter, they probably are looking for a somewhat large change and maybe a couple hundred or thousand good phosphosites will do it. If they're the former, do a big enrichment and, for good measure, run the samples on an ion trap -- BOOM! 7 million phosphopeptides from 60,000 MS2 spectra. 

(Kidding about a decent amount of that last paragraph.)

Okay, but for real, sometimes you are going to need phosphopeptide enrichment and there are crap ton of different strategies. What is the best use of your time? 

We've come full circle! 

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