SweetSeqer: Glycopeptide analysis software!

More magic from the Steen and Steen lab!  In press at MCP is this paper from Serang et al., that details a new software package called SweetSeqer.  This uses a de novo type approach to evaluate glycopeptides.  For any of you guys doing this, you know that the vast majority of the time we end up doing in manually.  In this paper they show a couple of spectra where an expert manually works through it compared to the results that SweetSeqer comes up with and it looks pretty good.
You can download the software at the Steen and Steen website here.
In order to install it, you'll need a couple of things.  The first is a python compiler, and the second is the pyteomics toolbox.  Don't worry, they are both free and available here.

Spec&SepNow a pretty App you can't log into

I got a notice from the Apple Genius thing that said spectroscopy/separationsNow.com had a new app.  This would work similar to Sparkplug, where it would provide me with abstracts that met one of many criteria that I set for upcoming literature from Wiley.  The icon is bright and colorful and suggests that this was really well made.  Then you try to log into it....
Try it, it's fun.  You get two identical bars.  The first says:  enter you registered user name.  The second says: confirm registered user name.  Then hit the login button.  Then it declares that the username or password are incorrect.  Try putting your registered password in the second box.  Doesn't work.  Try any combination until you realize that you are typing 2 really long things on a tablet and then delete the App.
Normally I only try to write nice things, but I would expect Wiley to do a little better.
If they fix the App, feel free to let me know.  It seems like a good idea, but I'll just keep using Sparkplug.

Korean HUPO 2013

The KHUPO conference is the 28th and 29th of this month in Seoul.  The highlights for me?  Top notch researchers like Akhilesh Pandey, Mike MacCoss, Michael Freeman, and William Hancock.  Another big one for me is Henry Rodriguez who will be describing the ProteoGenomic Atlas, a program at the NCI that I hadn't heard about until now.  Dr. Hancock will be talking about chromosome 7 and the group in Seoul that is working on chromosome 11 will detail their progress as both are working toward the comprehensive human proteome project I touched on a couple of weeks ago.  I'll also be there, talking about quantification technologies.  For more information, check out the KHUPO site here.

Xcalibur 64-bit

This doesn't come from the Xcalibur that I mean, it is just a little cooler looking.  It appears to be from some Casino gambling game.
This is just a quick blurb in case you didn't know that a 64-bit version of Xcalibur is available.  In December I spent a lot of time distributing the file to people but I worked with a group just last week that still didn't have it.  It is out there.  It is available. And I have yet to see a single glitch (particularly over running a virtual environment on your 64-bit PC to run the 32-bit version.)

Do you need nanospray to do proteomics?

I've been doing MS for quite a while.  Nanospray was definitely around when I started, but it wasn't the way that most labs did things.  I toyed around with it a little in grad school, hated the existing technology at the time, and didn't do it again until my second Postdoc.  I still kind of hated it.  Microflow electrospray has so many advantages:  the pumps are more precise, they are easier to set up, troubleshoot, they degas themselves, the clog less often, etc.,
Nanospray, however, has the distinct advantage of a 1 to 2 order of magnitude increase in sensitivity so we have to use it, right?
What if I said that I'm starting to see labs that are rejecting the use of nanospray and are going back to micro flow electrospray for proteomics and are generating very nice results?  Here is the argument:  When the first nanoflow experiments were being routinely done, say 10 years ago, this was a tremendous jump forward for these relatively insensitive instruments.  The increase in analyzer sensitivity on current instruments is far more than 2 orders of magnitude.  Is it 3 or 4 or 5?  In any case, from this line of thinking, the advantages of microflow may outweigh the disadvantages of sensitivity for some experiments.
The image above is from one such experiment.  In this experiment, a 3 uL injection of a BSA standard digest at a concentration of 2 pmol/uL was ran at 200uL/min on a 15cm C-18 column.  It is difficult to see from the screenshot, but the gradient was (in total) 34 minutes.  The base peak intensity is roughly 2 E 8.
The punch line?  This digest was ran on an Orbitrap XL that could have had further optimization for this experiment.  Yes, the injection is pretty high by most standards, but imagine this same injection on a fully optimized Orbitrap Elite or Q Exactive with a little slower gradient.  We would easily be seeing a base peak of 1 E 9 due to the relative increase insensitivity and I expect we could drop this injection by 1,000 fold and still fully resolve this protein.  Hypothetically, this doesn't sound like we'd be that far off from a normal experiment now, does it?
Just a thought, but I'm excited to see what will come of these labs that are switching over to higher flow rates.

First step in the human proteome project

Detailed in this month's JPR is the first step in the HUPO human proteome project initiative.  This extremely interesting paper from Gyorgy Mark-Varga, Gilbert S. Omenn, Young-Ki Paik and William S. Hancock describes the layout of the chromosome-centric steps of this project.
The paper also has one of my favorite figures of this year (stolen blatantly from the theHpp.org)

Unlike the figure above, the one appearing in the paper is completely filled out.  I LOVE that the chromosomes were divided between so many collaborating labs in so many nations.  Science, the great unifier!
With the completion of this project (projected to be 2022), I may have to back off of my criticism of certain semi-targeted techniques, such as DIA.  If we truly have a library of the proteins present, these technologies start to make a lot more sense.
This paper is a short lunch break read.  I highly recommend that you read it in order to see where our field is going!

ProtMax -- automated spectral counting!

ProtMax was mentioned in a new paper this month in MCP, and Google helped me find it.  It is a really quick, simple, and extremely useful application provided by the University of Vienna and written by Dr. V. Egelhofer.
You upload the processed MzXML file and it automatically gives you quan data by spectral counting.  It can take into account retention time and link your spectral counts to your protein.  You can also make it search a specific target list, rather than the full scan data.  At the site it is also possible to download test data to verify that your formats are correct.  Hopefully there will be instructions soon for this handy little tool!

Tandem metal oxide enrichment for phosphoproteomics

"Arabidopsis", a word that strikes boredom into the hearts of so many students throughout the world! Mostly kidding!  I know how important it is to have good model organisms.
As proof, look at this great new paper in MCP this month from Hoehenwarter, et al.,.  In this study they used a tandem metal oxide enrichment procedure to do a bang up job characterizing the MAP Kinase substrates in this silly little plant.  I didn't even know plants used MAP kinases, which are one of the very central signaling pathways in cancer and most diseases.
In the study they used aluminum oxide to enrich intact phosphoproteins, digested them, then enriched the phosphopeptides with titandium dioxide.  The resulting double enriched phosphos were analyzed on an Orbitrap XL.  On this super enriched sample, they even got quantification data by using spectral counting in Proteome Discoverer and a new (to me, and apparently not-yet-published) software package called ProtMax.  Of course, I can't wait to investigate this!

Is iTRAQ 8-plex ruining your experiments?

I have done a lot of iTRAQ.  In my last job we used the iTRAQ 8-plex kits as our most common experiment.  Imagine my surprise when I was sitting in a presentation where the weaknesses of the iTRAQ 8-plex kit appeared to be a given!
 While I'm still waiting on further clarification and possibly a follow-up meeting with the technical experts to assembled this publication, I'm doing some digging.  And what I've come up with is that in the last 9 months or so the iTRAQ 8-plex kit has become one of the most reagents in our field.
 There have been some extremely critical papers recently, such as this one from Evans et al., where they show that iTRAQ constantly underestimates the ratio fold change.  A discussion of the paper at Shared Proteomics suggests that this may have been an issue with the HCD fragmentation on the Orbitrap XL.
  The guys on the forum also brought up this paper from Pottiez et al., that appears to completely refute these findings.  They show that using a MALDI-TOF/TOF and studying known ratios experimentally that the iTRAQ 8-plex is considerably better than the 4-plex kits.
 Further searching led me to this presentation from Matrix Science (MASCOT), presented at ASMS 2010 that also delves into the under-representation of iTRAQ ratios as described in this 2009 paper from Yen Ow et al., that has a pretty shocking illustration on the front (below).

When I moved into iTRAQ after years of label free and SILAC studies, I read a lot of iTRAQ papers.  The fact is that it is such a popular technology that there are just a staggering number of them out there -- and it is impossible to read them all.  This is how I interpret all this stuff I've been reading the last week:  Every technology is going to have some controversy around it, but if people are still getting good results from that system, then we need to roll with it until something better comes around.  I think the data out there that says which system is better for iTRAQ essentially neutralizes itself out.  4-plex and 8-plex are each going to have their distinct advantages and disadvantages.  As for the ratio suppression issue in iTRAQ -- we aren't using this as a definitive filter.  When we see a 2.3 fold up-regulation we don't really, literally, believe that protein is up-regulated 2.3 fold, or that it is an inferior measurement to another protein that is up 2.8-fold.  We know that in quantitative proteomics we have a huge error bar on every measurement of a complex digest.  If we are using iTRAQ to implicate pathways and keep take those ratios with a "grain of salt", as I think we all are doing, 8-plex iTRAQ is currently the fastest and best way to screen multiple samples at once.
Now if something better comes around, it comes around, but for now, we're going to keep running our 8-plex iTRAQ data.

TraceFinder blog

This is a little off the topic of proteomics, but still on the topic of mass spectrometry, specifically for people interested in smaller molecule quan:  The TraceFinder blog is packed completely full of information on this software including new updates, video tutorials and, my favorite, the "Ask a Guru" option.  It would be hard to find a software package that is being supported as fully as Tracefinder