Friday, April 1, 2016

Plug n' play analysis....of the Human Phosphoproteome!?!?!? Study of the year!?!?!?

When I hear "plug and play" it makes me really happy. Remember when we had to get a CD (or floppy drive...and install software drivers) then plug in our new peripheral into a COM port and then go to the hardware settings and enable the stupid device? "Yay! A new controller, maybe I'll be able to use it to play DOOM in a half hour or so..."  Plug and play. Plug in the new mouse. Boom it works.

So....Plug N' Play human phosphoproteomics?!?!  What??  That's 2 awesome things, but that doesn't make sense. Phosphoproteomics is really hard. You've got to spend a week preparing special metal affinity columns and enriching samples and running the sample 50 different ways and on and on!


Check out this new paper in Nature Methods from Robert Lawrence et al.,!  They take a complex phosphoproteomic model (MCF-7 breast cancer cell line) and look at the phosphoproteome using normal (difficult and described above) shotgun DDA techniques, then DIA (like that SWATH thing), and finally with Parallel Reaction Monitoring (PRM) for targeted phosphopeptides of interest.

What did they find out? PRM kicks ass. Actually, PRM totally kicks ass. I swear, this is rapidly becoming my favorite thing. When they targeted 101 phosphopeptides that they were interested in -- some of which were decent abundance, some of them were exceptionally low abundance (DDA and DIA didn't even pick them up!) -- they found that not only could they detect them, but they were very reproducible.

If you're thinking "so what? we know PRM is more sensitive than DIA and DDA. When I do phosphoproteomics the traditional way, I come back with 40,000 phosphopeptides." Hey, I'm not knocking the peak bagging labs out there. If your goal is to get a method that gets more phosphopeptides than the last paper, more power to you. But a lot of biologists just care about one pathway and how much information they can get about that pathway in a run. And this is where this paper makes the turn that got it into a Nature publication

You can access this resource here.

Did you just read those three bullet points and run around laughing and jumping up and down? Okay, maybe that was just me. WHOA!! Seriously? This can't be real, right?  No way.  Sorry, another round of me laughing like a psycho and scaring my dogs!


Check this out!

You tell it what pathway you are interested in. In this case, I'm going to say AMPK signaling as an example. Then it'll grab my known phosphosites that are visible to mass spectrometry with tryptic digestion. I can pick individual ones...or I can say...grab the whole known pathway by clicking the top option (lets see how it works with a whole complex phosphorylation cascade...)

It plots everything over a 60 minute gradient. The vertical axis is the number of targets that should come out then (this is, btw, experimentally observed data!!!!). So, at 10 minutes in, 8 of these targets should be eluting at once. my QE classic running 35k resolution PRMs gets about 8Hz. Ummm..perfect!

If there are too many, I can eliminate phosphosites that are of less interst and it immediately re-adjusts. HOLY COW!  Wait, it gets better!

Once you get this all set up right, there is a button at the bottom that says "Export Schedule". What's that do? Well, it just automatically makes this method for your Q Exactive.

I'm stretching it a little. It makes an Excel file. You do still have to open your QE method software and hit CTRL+C and CTRL +P.

Lets sum this up:  Human cells respond to stressed by signaling down distinct and reasonably well-understood pathways. Understanding these pathways is critical to the diagnosis of many diseases and often even to determining what chemotherapies people get when they have cancer. And these guys just made it so that you can instantly make a method for analyzing any one of these pathways. You can be ready to run in 5 minutes or so.

Its only April 1st, but this is my early pick for paper of 2016. If you don't want to hear me go on and on about how awesome this work is, you should probably try to avoid me!

Coincidentally, all this enthusiasm is getting in the car with me (after I have a shower) and going with me to the National Cancer Institute this morning. Can I work it into every single conversation I have today? Damn straight, I can!

Sincere thank you to this team for this amazing work, and to Julian for tipping me off to it!!!!


  1. Hi Ben, how did you get the PRM box in the 'Experiments' section? My one doesn't have this option in the 'General' list on the left side.

    1. Howdy, Rambo! You probably just need to upgrade your Exactive Tune software. I think it appeared in Tune version 2.3. I'm a big fan of 2.5. You should be able to get it at:

  2. Too bad Pierce (aka Thermo) just discontinued their retention time standards :(

    1. Brett, really!?!?! I'll check on that. Are you sure they didn't just change the part numbers (I think everything is moving onto Life's servers). I'm hoping that isn't the case!

    2. Not 100% sure, but I've been told by a few people who tried to order it that it is unavailable.

  3. Thanks for the enthusiasm. Always been a huge fan of your blog.


    1. Thank you for reading! I'd still write this if no one was visiting the site (that was pretty much the case for years) but it is honestly cool that people enjoy my weird hobby.