Deep proteome coverage by 3 dimensional fractionation

Do you REALLY need proteomics depth?  I mean, do you REALLY need it?  They we know how to get it.  Fractionate fractionate fractionate.

This new paper from Ilian Atassov and Henning Urlaub shows how to get crazy deep proteome depth by employing a 3D separation approach.

1) Do SDS-page at the protein level
2) Cut band and digest
3) Separate the peptides by isoelectric focusing
4) Run those fractions on LC-MS/MS

Using this approach, they were able to identify >3,000 peptides out of a single gel slice.  Worth checking out if you really just have to get to the bottom of your proteome.

NeuCode for de novo peptide sequencing?

On Tuesday I saw my favorite talk of the year.  Anna Larson-Merrill from the Coon lab gave a talk on NeuCode proteomics for the Indianapolis NPI conference.  What an amazing and flexible technology!  All of the benefits of SILAC and TMT, with only the limitation that ultra-resolution is needed for quan.

Then I found out that yet another application of NeuCode has been discovered.  In press at MCP, is this paper from the Coon lab where they show the use of NeuCode doublets for the improvement of de novo sequencing data.  I stole this figure (below) cause it's too cool to not post (don't sue me! see disclaimer page!)

If you are interested in de novo you need to check this out!  (Bonus points for these authors for using pepNovo+!)

Updated TMT 10plex method in database

The Orbitrap methods database just had its first suggestion/correction.  The scientists of Thermo's Proteomics marketing division, Dr. Rosa Viner and Dr. Michael Blank have spent months optimizing methods for running the TMT 10plex experiments on various instruments and have provided me with an optimized method for this.  It is titled "revised" in the methods database.

Thank you!!!  Let's keep these comments and suggestions coming.  I really want to make this a working resource available for everyone.  Orbitrap methods database.

Hekate - A new crosslinking analysis program for people with experience in Linux and Perl

This new article (ASAP) at JPR describes Hekate, a new software package for evaluating protein crosslinking.  You can download it from GitHub here.

And thats where I have to stop.  I have Perl and when I really want to, I can run a Perl script.  Unfortunately, this is a tough one.  GitHub suggests that you get support from another resource (link provided).

 Hekate then falls on my list of nice software ideas that are implemented in such a way that the software really isn't accessible to labs that don't have their own bioinformatician or programmer.

New phosphoproteomics review in press at MCP

In press at MCP is this nice new phosphoproteomics review from Philippe Roux and Pierre Thibault.  While the first half of it is your run of the mill review on this topic, the second half is a very nice review of recent findings in cross-talk between various post translational modifications.  The figures are also extremely nice quality.  All in all, a pretty nice review and definitely deserving of placement in MCP.

Nice article on Next-gen sequencing.

Shotgun proteomics requires databases.  So-called "next gen" sequencing devices rapidly and (relatively) inexpensive new databases.  This article at IEEE Spectrum gives a very nice run down of how these new devices work, as well as a nice look at the problems we are facing with where to place all this data.  Since this is obviously a problem we're also seeing in our field, hopefully one group will come up with a good solution soon and we can share it!

Intact antibody ran on the Orbitrap Fusion

I heard a weird rumor the other day:  That the Fusion can't do intact protein analysis.  I disputed it when I heard it, and figured I ought to have these screenshots up somewhere in case this myth perpetuates somehow.

This is from a Fusion OT run on a commercial reduced antibody at 15k resolution.  I did a rough deconvolution and it came out pretty solid:

For this antibody, the expected masses are in the table above.  Not too bad, right?  Now, if you were being really critical, you'd probably say this isn't all that pretty.  And the truth is that 15k resolution isn't that amazing for an intact analysis -- fortunately this is an instrument that can do 450,000 resolution.  And this file wasn't optimized forever to look amazing.  It was just a run to see what the intact masses look like on the instrument, when it looked okay, we moved onto the next test.  If you have further doubt about the ability of the Fusion to do intacts, I can upload this RAW data file.  Please leave me a comment below if you want it and I'll make it available.

Encyclopedia of Proteome Dynamics

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The number of tools out there for our field is pretty amazing these days.  The Lamond lab at the University of Dundee has provided us with another.  Part of the PepTracker project, the Encyclopedia of Proteome Dynamics is an extensive tool to visualize large scale changes in quantitative proteomics.  I can't wait to spend some time on it!  I'll write more later after I check out the features, but from what I've seen so far it is a darned nice tool.  You can find it at PepTracker.com here.

p53 post translational modifications

I'm running a high fever and I already reached the end of the internet.  Time to go to MCP!  Let's get this blog running again.

If you are a biologist, chances are you are familiar with p53 (shown in the image above that I brazenly stole from Dr. Andrew Martin's lab)  That is because p53 is involved in all sorts of things.  Primarily, however, it binds to DNA in response to damage and either stops or slows it's progression through the cell cycle.  Many cancer cells are either deficient in p53 or have mutations in it or in proteins leading in its activation pathway somewhere.

The picture above (check out the link, it's cool) shows the frequency of mutations in p53, but that isn't what this article is about.

In press at MCP, is this very nice paper from Caroline deHart et al.,.  In it they detail a high resolution proteomics approach to characterize the PTMs of p53 under different conditions.

On a technical level, they used p53 pull downs, ran them out on gels, and ran the gel fractions on an Orbi XL or Orbi Velos using either stage tips or an Advion NanoMate.

And they found PTMs all over the place.  Phosphorylations, methylations, ubiquitin sites and on and on.  This is a really cool paper to really illustrate how complementary genomic and proteomic techniques can be, because (as shown in the picture) we know an awful lot about the various mutations in p53, but that won't get us the whole story.  Proteomics gets us another step closer.

Highly recommended for you cell cycle people (obviously) and my friends in the cancer realm.

DMSO proteomics study reproduced perfectly in my hands.

Wow.  It's been a busy month!  We haven't gotten around to posting anything.  I promise there are good reasons for this and I may be doing some backdating.  I've been wrapped up with all these amazing new instruments that are out this year.  I have got some really nice data.

First of all, however, is this DMSO study I talked about in my last (long long ago) entry.  In limited experiments before I was cut off by the U.S. Government shutdown, I found that the results reported in the paper were spot on.  3x increase in TIC intensity, 30% average decrease in fill times and a corresponding increase in peptide IDs.  This seems to carry over whether doing an unlabeled study or a TMT labeled analysis.  At first glance, my baseline seemed a little higher than I'm used to seeing but I do not have the data yet to support that.  Hopefully someone will be able to smuggle my RAW data files out of those poor deserted buildings and I'll get some JPEGS up.

Again, it is still uncertain what, if any, effects 5% DMSO will have on LC pumps, seals and/or MS components, but if you're willing to take that gamble, there is a great deal of sensitivity to be gained.