Analysis of phosphopeptide enrichment strategies

About a year ago I had a great conversation with a scientist from Cell Signaling who described the work they were doing with differential phosphopeptide enrichment. Now they have some figures up that describe the awesome work they've been doing!

If you are still using the FACE technique or a series of different enrichment strategies leading up to FACE you might want to take a step back and think about what you want to get out of your samples. Is a generic anti-phosphotyrosine antibody still the best for what you want out of your analysis? If you are interested in pathways, for example, that preferentially use phospho-Ser, maybe there is a better option now than we had 5 years ago!

Proteinaceous -- Where to get resources for Prosight!

If you are looking for information on top down proteomics via Prosight or info on the Prosight nodes for Proteome Discoverer, you need to check out Proteinaceous.net.

Here is a direct link in case you have as much trouble spelling it right as I did...

Nature Milestones mass spectrometry!

Shoutout to David Kusel for the link for this one! Is there anything about the history of mass spectrometry that you've ever wanted to know? This Nature Milestones project probably has it covered. This was compiled by a huge list of authors who all seem to know at least a little about this field and is written to be accessible to everyone.  It would make a great reference for our customers or collaborators who aren't really sure what magic we're doing in our noisy rooms!

LC-MS/MS applied to directly study DNA damage in Wilson's disease

(Image courtesy of Hendrik A. van Dijk & Herbert L. Fred, MD, original link here.)

This new paper in press from Yang Yu et al., at MCP is fascinating for a ton of reasons. First one, I have never heard of Wilson's disease and I had to read up on it in this Wikipedia article. In a nutshell, its a recessive genetic disease. If you get stuck with two of the copies because your traitless parents both had it then you accumulate excessive copper in your system. This copper messes with your liver and maybe your brain and it is somewhat subtle and very difficult to diagnose. Sometimes you have oddness in your eyes that is indicative, as shown above.

Another reason this is fascinating? They detail a painstaking method of directly analyzing DNA damage via LC-MS. The introduction of stable isotopes leads to an absolute quantification method via triple quad and ion trap mass spec. It is really a fascinating method because when we think DNA damage, we think about assessing downstream effects (got the right affect/effect this time, I think!). If I want to quantify DNA damage, I'm going with phospho-H2AX quantification or something like that. These guys cut out all the middlemen and go right to the DNA!

Biocrates -- QC'ed kits for metabolomics!

I'll not pretend to be a metabolomics expert, but its super interesting, right? In terms of sample prep, they have it far far worse than we do. At least we know how to get most proteins in one process. Metabolites? That's a different story.

Biocrates is a company that hopes to make MS based metabolomics easy. They produce QC'ed kits that are specifically focused on clinically interesting metabolites. You get the sample prep kit, the conditions for the experiment and the software to process the data. What you need is the mass spec -- and you are doing full out metabolomics!

Currently they kits are optimized for triple-quads but they are in the process of getting these powerful tools validated for the Q Exactives!

Washington and Baltimore mass spectrometry club

Due to some changes in what I do during the day I now get to spend a whole lot more time in the same state where my house and my dog live.  In my exploration of the area for fun things to do, I lucked into a last minute chance to go to the D.C.-Baltimore mass spec club!

If you are around the area you should check this out. The website is here.

I learned a bunch of stuff!  I  got to meet a senior scientist at AP-MALDI and I think I'm going to get the chance to set up a source and do some imaging mass spec on a Q Exactive!!!

If you're around the area anywhere you should come and check this fun group out. I can't imagine missing another one of the meetings!

Proteome Discoverer 2.0 / 2.1 workshop in Vancouver!

If you are going to beautiful Vancouver for International HUPO, you might want to pop by the Proteome Discoverer workshop where you'll get to see the introduction of this guy!

Wait? What? We just started using PD 2.0...are you crazy? Yes, but that's beside the point. PD 2.1 is a follow-up package that looks just like PD 2.0 but better. There were features and improvements that were recommended by all you users out there that just couldn't make the 2.0 cut.  Its so good that I pretty much just use PD 2.1 for everything.

Here are the details!!  This is meant to be interactive, not "DEATH BY POWERPOINT". Bring questions, data, whatever. This is great software and we want you to walk out of there with the ability to generate better data!

Here are the details I have right now. I'll add more info as I get it.

The effect of peptides/protein filters.

We're a field that loves to count things! As we've matured as a field the numbers have been a great benchmark for us. And they keep getting bigger all the time. Better sample prep, better separation technologies and faster, more sensitive instrumentation is making it possible to generate data in hours what used to take days or weeks not all that long ago.  

In papers where we detail these new methodologies, we see one type of protein count filter, and I think we see something a little different when we look at the application of these technologies. If you want to show off how cool your new method is, when you count up the number of proteins you found you are going to go with 1 peptide per protein for certainty. In my past labs we recognized those methods as great advances, but I sure had better have at least 2 good peptides before I justified ordering an antibody!

 I don't mean to add to the controversy, by any means. I think using one peptide per protein can be perfectly valid. Heck, we have to trust our single peptide hits when we're doing something like phosphoproteomics! Cause there's just one of them. And if I'm sending my observations downstream for pathway analysis, I'm gonna keep every data point available.  I just wanted to point out how the data changes.

I downloaded a really nice dataset the other day. Its from this Max Planck paper and uses the rocket-fast QE HF. I picked one of the best runs from the paper and ran it through my generic Proteome Discoverer 2.x workflow.

In 2 hours I get about 87,000 MS/MS spectra. If I set my peptide-protein so that any single peptide means a protein with this setup I get 5,472 proteins from this run.

Now, if I apply the filter 2 peptides per protein minimum...

Ouch!  I lose over 1,200 proteins!  

Are they any good?

Okay, this is an extreme outlier, but this protein is annotated in Uniprot, so its a real protein and it only has one peptide!  This is 92% coverage!  I didn't know there were entries this short in there.  If we went 2 peptides/protein we'd never ever see this one.

The best metric here probably is looking at FDR at the protein level.  (I did it the lazy target decoy way 1% high confidence/ 5% medium confidence filter)

Its interesting. Of these 1,200 single hit proteins, about 150 of them are red (so...below 95% confidence). Another 150 or so are yellow, but the rest ~900 proteins are scored as high confidence at the protein level.

Okay. I kind of went off the rails a little. Really, what I wanted to take away from this is how very much using a 2 peptide count filter can affect your protein counts.  The difference between identifying 5400 proteins and 4200? Thats a big deal and worth keeping in mind. Is your data going to be more confident if you require this filter? Sure. Are you losing some good hits? Sure, but its your experiment and you should get the data out at the level of confidence that you want it!

pVIEW: tons of tools, including 15N (n15) quantification

I swear I wrote a blog post on this years ago. Seriously, but it took me forever to find this software and then re-remember how to use it.

pVIEW is a really nice piece of software. It does a ton of different things...including 15N quantification!

It is incredibly user-friendly. If you are using a Thermo Instrument, I highly recommend you download this tool as well:

Whats it do? Well, you click on it and show it a directory. And then, without complaining or without any extra steps it converts you data rapidly and perfectly to mZxmL (not sure I capitalized the right stuff.) Then you can pull your data right into pVIEW.

pVIEW can be downloaded at the Princeton Proteomics and Mass Spec core website here.

Run complete programs on any system with BioDocker! Wait...what's a BioDocker?

Okay. I'm going to be pretty excited about this whole thing, cause I knew about exactly none of this 20 minutes ago.

It is totally awesome that we have all these talented programmers and bioinformaticians out there writing interesting new code. A problem is that, just like any expert in anything, they start talking their expert-ese and it becomes hard for outsiders to figure out what they are talking about. I take things about proteomics -terms and such, for granted all the time even though I try very hard not to.

This is an acknowledged problem in their field. That they can't reach users cause sometimes users don't know what a Perl thingy is.  Even worse, maybe someone assumes that you have that Perl thing on your PC because they've had it on every PC they've owned in the last 15 years.

An awesome effort is underway and its called Docker. Its generic for everybody, but what I can understand of it is that its a "container" for a program that includes all the requirements for running it. Say you need that Perl thing and some Perl add-in things, then it would be included in the Docker.

A more focused thing for us is BioDocker. Same goal, but specifically for bioinformatics type stuff.  Sounds great, right?!?

BTW, I'm learning all this from Yasset's blog.

Cause you know what? They've already constructed two awesome proteomics BioDockers.  The first is the all-powerful Trans Proteomics Pipeline and the second is the DIA-Umpire!

Is it simple enough that a dummy like me can use it? Actually...I think it might be...not without challenges, but its getting there!

If it isn't 100% what we need/want right now, its a great step in the right direction. Lets get all these awesome tools and put them into an easily digestible format. They get more users which hopefully translates into more grant justifications and more cool algorithms and we get better data!  Win win win!