Caspases can be regulated by protein phosphorylation

Caspases are proteins that chew things up.  If you didn't know better, they look like they chew things up completely indiscriminately -- every protein gets chopped up.  This is really useful for controlling diseased cells during things like apoptosis.  This cell is bad, program cell death, destroy everything.

This awesome new paper in this month's MCP from Jacob Turowec et al., (permanently open access!) shows that caspase cleavage may not be indiscriminate at all.  In fact, it may be regulated, while it is happening, by protein phosphorylation.

To decipher this, the team employed an unbiased n-terminal approach they call TAILS and analyzed the rate of degradation of proteins in caspase activated systems.  The analysis was performed on an Orbitrap Velos and the data was analyzed with MaxQuant.

They find that some phosphorylation events lead to increased cleavage rates and some lead to decreased rates.  Interesting, right!  It seemed strange that we would just auto-self destruct cells indiscriminately.  Even in this worst case scenario, it appears that we have some measure of control after all.

I stole this image from their paper because its cool and describes their aproach.

Free training courses from EMBL

There just isn't a good excuse these days to not know stuff. Its too easy to learn about anything you want!

For example, wanna take free classes in:

Metabolomics
Next gen sequencing
Genomics
Protein interactions
PROTEOMICS
or
Reactomics (whatever that is?)
and so on..and so on...

Follow this link to EMBL and check it out!

To deplete or not to deplete...part 7 or something....

Plasma protein depletion?  So many opinions out there!  How about another one?  How about the most cited paper from Open Proteomics!?!

Proteomics of human plasma: A critical comparison of analytical workflows in terms of effort, throughput and
outcome, is that paper.  (Open access! Wooo!)

In this one, the authors take a serious run at the pluses and minuses of depletion (like what a HUGE protein loss they take with depletion...quantified! from different depletion techniques).  Then they take the depleted, undepleted, etc., and run it all out on an Orbitrap Elite using the same settings.

The results?  Well, thats a surprise.  Its open access and they made some nice Venny Venn Diagrams.  Worth checking out! (and easier to take seriously than David Tennant doing Hamlet!)

HPRD -- A protein database from the Pandey lab

I've known about this one for quite a while, but I just realized I don't have anything up on the new blog (and I never fully transferred everything I've written from the old one...and probably won't at this point, lol!)

The HPRD is another cool protein database that is a joint effort from the Pandey lab and the Institute of Bioinformatics.

Check out the output here on ITGA5:

Quick reference info at the top:  class, GO function and process:  followed by a quick graphical breakdown of what we know of the protein at this point.

Followed up with info on pseudonyms for the protein, substrates, etc.,

You have other options as well, such as looking up canonical pathways.  In a minor criticism, the pathways come up in HTML as a list and must be downloaded for viewing/linking.  I definitely prefer when KEGG or ball and stick pathway models pop up, but I can get those from other sites.  Those other sites won't give me graphical breakdowns of my protein domains, and I think this is the real advantage of this site over other ones out there.

You can link directly to the HPRD here.

Moped Expression database

When I hear the word "moped" I think of the image above.  This was the bike I wrecked had in grad school.  Fun and insanely easy to wreck do tricks on.

Its pretty cool to be able to associate this word with something less painful.  The Moped expression database is one such thing.  Here is a screenshot.  It looks like a fantastic resource!

I popped in one of my favorite proteins, ITGA5, and I immediately get expression ratios of this protein in known cell lines.  Is it upregulated in my favorite cancers?  You can't see it in the screenshot, but there is a column off to the right of the screen that hyperlinks me to the experiment(s) that made these observations!

That proteomics data suggesting some differential regulations that you find surprising?  Pop it in here and see if anyone has ran into it under a different context!   You can even filter your observations by relative or absolute protein expression levels.  Sounds applicable to our field!

Credit goes to Alexis for finding me yet another cool resource to share!  The Moped database is here!

Scorpion Venom!

Soooo....I'm imagining the thinking here:  I have a mass spec.  What is the most badass thing I can do with it?  What is more badass than sequencing Scorpion venom?  Nothing.  Sorry.

This masterpiece of awesomeness comes from Stephean Schaffrath and Reinhard Predel and is in this month's Open Proteomics.  In case you were wondering if they realized how cool this was, just check out the image they submitted:

I can't exactly tell what is happening but I think that I'm looking at a Scorpion wearing handcuffs just hanging out by a mass spec.  I'm not sure.

And they went out of their way to justify this.  Not just: "this is so awesome, we have to do it".  No, they say that understanding scorpion venom might help us develop antidotes.  Bingo!

Now, they do use a MALDI, but they are doing simple barcoding.  And its on Scorpion venom, so they get a pass for just about anything.

For an encore?  They do some scorpion arm wrestling!  Nope.  Made that part up.  In fact, I was sure that was a thing -- even a scene in Over the Top (Stallone arm wrestling where the loser gets stung by a scorpion).

And I remember an episode of the A-team where Mr. T did it.  I may be making it up, cause Google is treating me like I'm a crazy person and not coming up with any images.

Okay, so this study is cool.  And unique (so far as I know).  If you are looking to get a redneck kid interested in science, this is exactly the kind of story you should tell him/her about.  Also recommended for anyone who is interested in peptide fingerprinting (barcoding).  Remember, these technologies are still around, and still good for all sorts of things.  Definitely check this paper out...or...

"Get over here!"
Last stupid Scorpion reference, I promise.

PTMScan Motif enrichment kits

At iORBI Boston yesterday I had a lot of conversations with cool people doing even cooler things.  One thing that stood out and I had to look up today was these new motif enrichment kits from Cell Signaling.

This is similar to the FACE (filter aided capture and elution) method that lots of people use these days for phosphotyrosine enrichment, but Cell Signaling has went a couple steps forward with this idea.  By developing antibodies versus specific known phosphorylation motifs (such as the recognition site for ATM/ATR!!! woooo!!!) they can pull down proteins that are phosphorylated specifically by proteins of interest.

Even better?  Kits are available that can target whole pathway!  Pick a pathway, enrich for the proteins containing your phospho motifs then DDA or target for your known peptides.

The Kinome kit contains ~20 different antibodies!  Direct link here.

Absolute proteome and phosphoproteome dynamics

I've been doing this stuff for a long time now.  And I'm starting to feel like I'm pretty good at it.  Once in a while, however, I read a paper that makes me feel like I'm still a bit of an amateur.

There is one in press right now at MCP.  This comes from Alejandro et. al., out of Boris Macek's lab and you can find it (currently open access!) here.

Strategy?  Let's start with a complex multiplex SILAC labeling scheme with an internal control (woo!) and do quantitative proteomics on it.  Then let's follow that up with phosphoproteomics of the same sample  And lets take the relative quan data from the SILAC, standardize it with our internal control and then compare the whole quan to the phospho at multiple stages across the yeast life cycle.

Whole proteomics was done with an Orbi Velos and Elite. The phospho was performed with an Orbi XL running CID neutral loss MS3.  Yes, I know, its another yeast study.  But take this one as a proof of concept paper.  We know a lot of these phospho events that we are seeing and when these observations line up it shows that we can get this amazing degree of quantitative certainty using only mass spectrometry.

Awesome study and great post acquisition analysis.  Strongly recommended, phospho people!

Back to serious business:

Abird -- little box that removes background ions

Apparently, people in Boston like to blow air across the fronts of their instruments.  Unlike the little device in yesterday's post which blows away all of the ions that are produced during the crappy parts of your run, this little thing, the Abird, blows a slow supply of clean air into your source at all times.

And it looks like it helps!  Y'all probably have known about this for a long time, but its new to me.

You can find out more about Abird at ESIsourceSolutions.com.

Want to keep your Orbitrap clean longer?

This is just awesome.  Maybe other people are doing this, but I've never seen it.

Can you see what that is?  It is a way of diverting the sweep gas so it is useful for nanospray applications!  At the beginning and end of a run, alternative tune files are used at BRIMS.  These tune files contain instructions to turn on the sweep gas.  This appears to effectively blow away the stuff that comes off the column at the beginning and end of the runs and extends the amount of time they have between cleanings!

Want to set your own up?

It's the P-430 Union at Upchurch Scientific.  This setup has been shown to work on both LTQ Orbitrap systems and the Q Exactive.

Correction (thanks guys!) its a P432, not P430.  For more details see this new post.