Study peptide methylation without antibody enrichment!

Keyun Wang et al., describe a super interesting methodology for getting to protein methylation sites in this new paper in JPR.

It takes advantage of two things:
1) That trypsin is going to pass right over a methylated lysine or arginine (the latter I wasn't sure about till I read this!) and
2) The methylation at these sites does not alter the overall charge of the peptide (this biologist didn't know this at all!)

SO....(ellipsis in case the next steps are obvious to any chemists reading this! Eureka?) they develop a chemical approach to exploit this and separate the bigger methylated peptides from the smaller non-methylated ones.

Best parts? (I like lists today!)
1) No derivatization!
2) Simple one-stage separation and it is seriously easy enough looking that I could definitely probably pull it off
3) It looks like it works great.

Further details: It appears to be basically high pH SCX (see what I did there?) and they get over 800 unique methylated peptides yanked out of a cancer cell line. I'm rushing through this one this morning, but they do the SCX on a tip (yeah!) and they use a SILAC-based approach to get some quan out of it.

Now, a critic might say that with some of the modern anti-methylation antibody approaches people have scored higher numbers of unique methylation events, but with this technique they don't see a bias toward particular peptide sequences!

Shoutout to PastelBio for 15 good papers I could add to my reading queue this morning!

Proteome-scale binary interactomics in human cells

(Image borrowed from: http://www.crl-mappit.be/contact/)

Ummm...in the clear winner of "Wow..Ben's knowledge of molecular assays is super crazy out of date and here is proof" contest, (maybe its time for me to read a book or go back to school!) I'd like to present the following paper currently in press at MCP.

Deep breath. Okay. So I couldn't even get through the second paragraph of the introduction before I had to start searching references. If you are interested in the most complex version of a Yeast 2-hybrid protein-protein interaction assay that anyone ever dreamed up, I suggest you check out: http://www.crl-mappit.be/mappit_toolbox/mappit_concept/ where I stole the first image of this post.

This methodology utilizes the well-characterized (but completely understood??)  JAK/STAT pathway to verify protein-protein interaction. And in the paper above, they used this, as well as a variation of this technique (called MASPIT) to interrogate a large portion of the interactome of Hek293T cancer cells.

Full disclosure: I'm pretty sure that even after having this open on my desktop for 2 days and looking at it...several...times, I still don't get it.

Fortunately! for my fragile sense of self worth I do understand how another Interactome project works cause it is like this:

This, of course, is the Harvard BioPlex Project that was described in this Cell Paper last year, is still going strong, and have even more data than it did when the paper came out. This, in my limited understanding, is the ultimate resource for what protein interacts with what.

Of course -- my first question when checking out this massively complex method for semi-globally checking out the interactome -- how do the results compare to the 50k protein-protein interactions predicted by BioPlex?!?!

And...they don't say. Heck, they don't even reference the paper!  Maybe they submitted it before BioPlex dropped this awesome resource on us. If you compare the results from these two techniques, definitely shoot me an email. I'm super curious now!

TKO TMT Standard for assessing isolation interference!

Isobaric tagging things like TMT and iTRAQ suffer from "isolation interference" it is a consequence of the technology. You can mitigate it by improving the resolution of your chromatography, with apex peak selection and by using MS3 based techniques.

Post processing software can give you a decent metric of your co-isolation interference, like this screenshot from PD 2.1...

(here this is a TMT10plex 2D separation I did a while back. This metric takes the total signal intensity of your selected ion divided by the total signal intensity of the MS1 window)  and this is a useful metric.

Joao A. Paulo et al., suggest an alternative methodology.   They describe how to create a standard by using deletion strains you can purchase from the ATCC that are described in the Yeast Deletion Collection.

The strains they choose have proteins that are completely absent, so if everything was perfect you'd get zero signal from all the peptides from these missing proteins in the appropriate channel!

I could see this being used a couple ways:
1) To optimize your parameters till you had parameters as close to zero signal as you could possibly get
2) To use as a pre- or post- run control to have the best understanding of how much you can trust your actual experiments!

An oddly satisfying reviewish paper on MultiOmics

MultiOmics might be my favorite word right now. I'm not the only one who is digging it. Lots of papers seem to have this new word in the title right now. And with good reason!  We've got so many ways of globally profiling what is in the cell that the next trick is going to be integrating all of them!

For an oddly satisfying review of the topic, you should check out this open access study in Nature Communications.  In the blog title, I'm going to leave the term "oddly satisfying." I'm not sure what it is -- is it the way this article rapidly tears through all sorts of -omics things? Is it the nice bold lines used for the illustrations that remind me of an Archer cartoon? Is it the cold medicine that has been slowing my brain down? I'm not entirely sure. What am I writing?

What I do know for certain is that, while this is an original study on the multiomics of E. coli it reads a whole lot like a review, cause it knows it has to introduce the reader to at least one -- if not many, of the things they're looking at. It also reads that way because this is almost entirely a meta-analysis of data from repositories. They do appear to do some of their own transcriptomics. Cause, you know, nobody's ever done RNASeq on E. coli before (though, more likely because it was easier to run their own samples than to download someone else's 2TB or RAW data).

In the end, though, the best part is that it shows how all these technologies are complementary. They can detect problems in their bacterial test system from data at the protein and protein structural level as well as in the fluxomics and at some of the transcriptional stuff. Key points here -- sure you get extra info from all these other techniques -- but it doesn't mean you have to do them to get to your answer!  There are many ways to get to the same answers!

Ever tried short tryptic digestions and failed? Try Partial FASP!

This brand new paper in Nature Chemical Biology  probably isn't there cause its got a really cool (and reproducible!!!) method for partial tryptic digestion. It probably helped, but it got there cause it conclusively identifies a new post-translational modification that can occur in histones and that has all sorts of sweet biological relevance.

But I'm a methods nerd and this is something I've tried to do -- lots of people I know have tried to do -- and I've never seen it work reproducibly well!

And theirs works!!!

They had to do it cause the PTM they are looking for falls into a domain where the trypsin would cut it up. And the method is really straight forward (and FAST!) and it makes peptides that shouldn't be ETD compatible --- big and ETD compatible!!!

I'll probably write more about this paper, cause I'm fascinated by the PTM, but its Halloween Friday. Check out this great paper!

In vivo protein structural information!?!?!?

We had an AWESOME meeting in Bethesda yesterday. A highlight of my weird job is that I have input into picking speakers for some meetings. The downside is that with 2 rooms running I couldn't see every talk.

This is one thing that blew my little mind AND I know I can talk about it cause it was recently published in ACS here!

I've got a minor obsession with what you can do with mass spectrometry and fast photo-oxidation of proteins (FPOP). 1) Cause its super cool and 2) I don't know how to run an NMR. But...in the end you can probably learn more about an in vitro protein or protein mixture from sending it to the weirdos who run the NMRs down the hall.

But...this...study blows the doors off of the little my brain has stored on protein 3D structures and interactions. This group shows that they can separate cells -- individually -- and then FPOP them -- and get information on the inside and outside features of the proteins --- from their in-cell environment!!

WHAT?!?!  I know!

This is a method development paper, obviously, but think of the implications here -- if you could get enough coverage -- you could have info on how your mutation affects the protein structure and even the protein-protein interactions of any of the proteins in that cell!  There are obviously some challenges -- huge search space and MS/MS sensitivity among others...but WOW! Right?!?!

Widespread K methylation in plasmodium -- and other things!

My Destroyer is getting set to reanalyze some old data while I'm out of the office today, thanks to this new paper.

In this, they do some really intense antibody-based pull-downs specifically looking for lysine methylations. And find them...all over...in Plasmodium falciparum. I'm always hunting for explanations for the spectra that we can never seem to explain from these organisms, and maybe some of them are thanks to methylations all over the place.  My first thought is throw in lysine methylation and just see -- then my next obvious thought is...is this inhibiting trypsin...?

So...in trying to hunt down some info on the topic...I find this paper...

...that strongly implies that maybe I ought to be doing meta-analyses on other proteomics datasets cause lysine methylations may be involved in all sorts of things outside of where we expect them to be (histones!)

One of the things that comes out of this Tuesday morning post-espresso paper binge is this fact: lysine methylation has been predicted the 4th most common PTM in nature? What?  Who decided that?!?

Turns out these guys did back in 2011!!

Check out this chart!! (Click to expand if you need to)

They looked at how often we observe PTMs compared to how often PTMs ought to occur. We know we're Phospho-biased, but it popping up as number 2 makes it seem like not such a bad bias. (I wonder how this chart looks now, considering how the glycoproteomics field has blown up the last few years?)

Does this post make any sense?  I know it lost linearity somewhere. What the frack is a geranylation?!?!?  I'd better hit the "Publish" button before this gets any more chaotic!

The phospho kiss of death!

I stole the idea for this post title from someone else, but its totally fitting for both the paper and for Halloween week!

You know how we normally figure out how a mechanism works in something really simple -- like a bacteria, and then we figure out how more complex organisms like us do things? This new paper in Nature from D.B. Trentini et al., shows us 2 super cool things:

1) Sometimes we figure out how the more complex organisms work first and
2) What the heck Arginine phosphorylation does!!  (And this is why it is in Nature!)

We have to break down and get rid of old proteins. Or our cells will all just end up completely packed full of old proteins (sub-ideal). In us, that is one of the things that ubiquitination does. It marks old proteins for degradation so we can get them out of there.

Until now, there wasn't a clear understanding of how this works in prokaryotes. Turns out...it is mediated by arginine phosphorylation (at least in the awesome model gram positive Bacillus subtilis!)

They work out here that a protease called Clp (fitting?) selectively finds proteins with Arginine phosphorylation sites and eats them up. They find it by doing Clp pull-downs of normal and heat shocked cultures of the bacteria and then back it up by elegant in vitro assays as well as with knockout strains of bacteria. You couldn't be more thorough.

Interesting note here for us lab rats --

--can I actually post this? (Nature, please don't sue me, if this is a problem please see my contact info under disclaimers and I'll take it down!)

What is highlighted above -- I don't know how to do, but I think its awesome!  Now that there are nanoflow UV detectors, does this mean I can rapidly determine during my runs my approximate tryptic digestion efficiency??  This bears further examination.

The phosphoproteomics is done with CID/ETD on an LTQ Orbitrap Velos Pro. (Unless there are 10 of them at IMP, this might be coming up on being the most famous Velos Pro in the world -- what a work horse!)

Anyway...super solid paper that absolutely deserves to be where it was published!!!

Mass spectrum jewelry!

This shop makes 3D printed mass spectrum bracelets and handmade metal spectrum pendants! What?!?! I know!

Opening Proteome Discoverer 1.4 Results in PD 2.1

Great question from a reader! Figured it deserved its own complete post while I'm in-between meetings!

You've got all those awesome MSF files you've processed in PD 1.4!  How do you open them in PD 2.1?  You definitely don't need to reprocess them all!!

Do this!

1) Make a new folder

2) Don't add any RAW files or templates or anything. Just blank on all that stuff

3) Where you add your RAW files -- add the PD 1.4 MSF files!

PD 2.1 should recognize all the stuff. Here it recognized that these 2 processed TMT fractions are -- TMT fractions and that I used the TMT 10plex. Now...I do have the RAW files in the same place where these .MSF files are. I'm not 100% sure if this is essential or not. Its just how my storage drive is organized!

Go to your analysis results and they'll be there as well!  w00t!

Now is the conversion part!  Highlight and Reprocess your data as a new MultiConsensus report!

Then make a Consensus workflow. I just chose one of the common default workflows.

Run it!!!!

TAAAADDAAA!!!