New methods in Orbitrap methods database

Recently, I've gotten to be in on a couple of Orbitrap Fusion installs and got to play around a little with different samples.  I've uploaded some methods that have produced nice results to the Orbitrap methods database.  The first is an MS2 only based method for iTRAQ and the second is the same for the TMT 10plex reagent.

In addition, I received some RAW data from a lipid researcher and uploaded his method for looking at lipids on the Q Exactive.  As always, treat these as a starting point and email me (orsburn@vt.edu) if you have questions/comments/suggestions. These are constructed in my free time and not meant to be considered as the end all, but for at least these 2 Fusion runs I'm pretty happy with what I got out of them!

READ MORE - New methods in Orbitrap methods database

Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma.

Mesothelioma is a cancer that starts in the protective lining around your organs.  Since the massive removal of asbestos in North America over the last 20 years or so, we rarely hear about this cancer now, as it was the major cause.  Asbestos was pretty useful stuff and since removal is expensive, there are lots of places where this cancer is still a big deal.  Unfortunately, there aren't good biomarkers for clinical assays.

Until now!  In this study, a team of Swedish researchers in conjunction with a medical institute in Turkey track down a series of good biomarkers for this nasty disease.  The experimental method was simple.  They used iTRAQ 8-plex to label (top-14 depleted) pleural fluid from Turkish patients with various lung conditions.  They used hi-resolution isoelectric focusing (see, the OFFGEL isn't dead!) to pre-fractionate the samples before running them on an Orbitrap Velos. 

Note on the method:  The method employed is one that could be considered suboptimal.  There are several places where the fill times and method could be tweaked to improve the theoretical peptide coverage and quantitative accuracy.  For current recommendations on running iTRAQ samples on an Orbitrap Velos, please see the Orbitrap methods database in the right hand of this page.

The results here, however, are extremely nice.  Multiple biomarkers were identified and validated with statistical significance which can only lead to an improvement in the correct profiling of the patients afflicted with these diseases.

READ MORE - Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma.

Most active Twitterers in Proteomics

Oh, Lists! Why do we love you so much?

In another example, Google+ suggested this article for me regarding the most active Twitterers (I don't think that is the best diction, but you probably know what I mean) in our field.  I follow most of these people, but will probably start following more.  I ran low on topics to write about over the holidays!

You can find the list here!

READ MORE - Most active Twitterers in Proteomics

Backdated --- Merry Xmas!!!

I took a break from the blog for the holiday but this picture is too awesome to not use it!

READ MORE - Backdated --- Merry Xmas!!!

Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo

Rad53 is a protein that is involved in DNA repair in yeast.  An extremely similar protein in humans, Rad51, is shown above.  You irradiate some cells and probe with an anti-Rad51 antibody and you get the distinct foci shown in green above.

Despite years of research, DNA break repair is something that is poorly understood.  A really nice paper in MCP uses a spiked in SILAC approach and high res mass spec to try to pull back the curtains.

In this multi-lab (heck, multi-nation) study, this team uses Rad53 deficient yeast strains and MMS (methyl methanesulfonate, a chemical that induces DNA double strand damage) to try to fish out the pathway leading to Rad51 activation.

Minor comment on the paper.  The MS1 search tolerance was set at 25ppm in the MaxQuant/Andromeda runs.  Particularly in a SILAC study, I think that this window is a little too big and might lead to mismatched pairs and maybe a raised FDR.  Otherwise, this paper is a nice solid look at using a good classic genetics (knockout) approach coupled with HR-MS to fish out pathway differences.  This is also one of those rare systems approaches successfully using spiked in SILAC.

You can pull up the original paper while it's still open access here.

READ MORE - Use of quantitative mass spectrometric analysis to elucidate the mechanisms of phospho-priming and auto-activation of the checkpoint kinase Rad53 in vivo

Mass spec terminology translator V1 is up

I have no idea what is happening in the image above.  GoogleImages gave it to me when I said "pug translator".
Anyway!  As long promised I finally put up the first version of the mass spec terminology translator (see top right).  This will be evolving.  I had a 6 hour flight with no internet yesterday and this was the first list I came up with.  As new jargon (and more importantly, jargon that I just haven't thought about yet) pops up I'll continue to add on, and hopefully clean up.  A bunch just popped into my head while I was writing this.  Expect expansion.

And a direct link here.

READ MORE - Mass spec terminology translator V1 is up

Mass spec free proteomics!?!?!

Lets start off like this:  This is a paper in MCP but they didnt do any mass spectrometry, not even a little, so why do we care?

Next article please!

No, wait!  This is awesome, I promise.

You know how I'm always preaching that the database ought to match the size of the thing we're doing MS/MS on?  Why on earth would we go to all the work of cutting a liver out of something, doing proteomics on it, and then searching all of the proteins that mice can produce?  Does that make sense?  Nope!  But there isn't much we can do about it.

But what if somebody took the genome and cut it down to what proteins are expressed in what tissues by doing RNASeq on individual tissues?

And what if they backed it up with protein arrays to show that the protein levels strongly correlate with the transript levels?

In my (humble?) opinion, thats a paper that ought to be in MCP!

Who needs a mass spec?  Wait! What?  Everybody!  But this is pretty cool and will be helpful later!!  Lets try re-running that brain, liver, or whatever with a FASTA that only contains proteins from that organ and see how our results jump through the roof and our FDR drops through the floor!

READ MORE - Mass spec free proteomics!?!?!