psims - A DECLARATIVE writer for mzML and mzIdentML!

As proteomics moves closer to the ideals of unified data formats (in both processed and unprocessed forms) we need to make sure every pipeline can support these formats.

psims is a great new resource for the weirdos who are using R and Python (!! joking!! just jealous of your neuroplasticity!!) to get into the uniform format bandwagon.

You can read about it (early/open) at MCP here!

If you don't want to read and just want the code, you can get it at GitHub here.

What makes it "declarative"? I don't know, but if the title didn't make you think of this, it will after you watch it.

A nucleic acid MS/MS search engine!!!!

Do you guys know how most labs check the accuracy of their PCR products?

By running gels....

It looks super precise -- until you realize that 100 base pairs is like 40kDa. You can get higher resolution (longer) gels, of course -- and there are other capillary based technologies -- but these things just generate an average mass. They don't tell you if something is wrong.

If only we had a NUCLEIC ACID SEARCH ENGINE!  We could use mass spectrometry to get a precise mass -- and sequence things! But that's more futurist mumbling, right?

Starting with BioPharma Finder 2.0 -- nucleotide deconvolution is a feature -- I was thrilled to find it and to see support in some other software as well. That's a good sign -- but a Nucleic Acid Search Engine is a critical next step.

If you'd asked me yesterday -- I'd guess 3-5 years and we'd see one...fortunately, I'm wrong a lot!

Consider how old-fashioned mass measurements are of amplified and often purified nucleotide sequences.

This group jumps a light year ahead and looks at how REAL RNA SEQUENCES are not only aligned -- but modified in an epigenetic sense.

Using HCD OT fragmentation of transfer RNA from people, they find they can monitor at least 20 different modifications -- effectively blowing open the doors on an entirely new way of studying epigenetics -- and maybe opening a completely new field for mass spectrometry!?!?!

I'm not sure the importance of this preprint can be overstated. This could revolutionize molecular biology and our understandings of the control between transcription and translation. And the method is straight-forward -- and the software is available for download here. Right now.

Glyconnect -- the next big step in the maturation of glycoproteomics!

Okay -- this is a big step in the right direction. We're getting much much better at glycoproteomics as a field. I have a figure somewhere that shows how many studies y'all are knocking out year to year... seomewhere... and this is the fastest growing area. What we need next? This.

Which you can read about here! 

This could be the UniProt for glycoproteomics. The site is obviously well-designed and ready for incorporating tons of new data.

What it needs -- is tons of new data. Get on it, everyone!! 

3D Print custom labware!

I scored my niece a 3D printer for Xmas last year. Black Friday thing and a lower price than I'd ever seen one for. Unfortunately -- a little too hard for her, especially after the company went out of business and it had to be operated with a series of open source software in order to work.

After replacing it with a more kid-friendly App driven alternative, I end up with my own 3D printer. You can only print so many Daleks....okay....well...you can print a lot of Daleks....but these things can actually be useful!

Did you know this existed?!?!  I didn't until just last week!

There is LOADs of custom labwear that you can easily download and print with glow-in-the-dark filament if you want!

A site called the "Thingiverse" is also loaded with great ideas like these.

I love all 3 of these. They're things designed to help those of us with freakishly large hands work with tiny PCR tubes.

There are some remarkable similarities between using the 3D printers and mass spectrometry. Resolution is a big deal. Expensive printers can achieve higher resolution in shorter time. Little discontinued $150 printers like mine can produce high resolution toys and tools, but it might take all night to do it.

If you've actually read this far and have access to one of these things -- this is the stuff  I've found the most user friendly:

Ultimaker Cura -- free software for taking premade objects and scaling them and adjusting them to print on anything

TinkerCad -- Create your own stuff for printing (which I then send to Cura and then convert to .gcode for printing)

Okay -- and just a reminder that THIS exists....

The flippening?

This has been coming for quite a while, right, but it still seems crazy. The Q Exactive hasn't been out all that long in comparison to our field -- but here we are. At some point since I last looked (can't find the post....but it didn't seem that long ago....) the Q Exactives overtook the great Orbi Velos for the most publicly available proteomics data sets.

And, at the rate that things are being published and dropped -- it won't be long before the QEs surprass all LTQ based Orbitrap systems.  The Elite is listed separate and is around 500 -- roughly where the Fusion systems are currently.

You can check out the interactive GUI for this on the front page of ProteomeXchange here. 

Optimizing MULTI-BATCH TMT!

Okay -- so TMT is great if you're comparing 11 things, right? But -- WOW -- does it ever get less fun to use when you've got 12 samples. Or 34.

There are many ways to tackle this, and these different ways have varying degrees of success. When in a pinch, I go to my dumb method, but it's VERY clear to me that this is crude and I'd love to have a better way.

THIS.IS.A.BETTER.WAY

24 (TWENTY-FOUR?!?!?) TMT 10-plex batches.

Coisolation explored.
Label isotope contamination explored.
Did y'all know that there is an entire chromosome(!??!) that roughly 50% of the human population has that the other half does not?!??
They use this evidence to determine the biological FDR. Cause...cells from humans that lack that chromosome don't have genes to make those proteins. If they show up in cells that don't have the genes, your FDR is probably askew....

The data is processed in MaxQuant. The post MaxQuant analysis was done with a bunch of R things, but the basic workflow is explained very well in nice figures and I feel like I can follow this logic to improve the next multi-batch set I try to combine.

EuPA YPIC problem solved and published!

This challenge was such an awesome idea! And I'm thrilled to see that it was tackled and solved.

You can read about it here. 

TOMAHAQ Companion! Targeted mutliplexed proteomics for everyone!

Hey! Remember TOMAHAQ?!? It's the technique that Gygi lab developed for using internally triggered targeting for quan, kinda like IS-PRM, but then the thing you target is actually TMT labeled?!?

You can download the original TOMAHAQ paper from Molecular Cell here.

Okay -- so TOMAHAQ is suuuper smart. Unfortunately -- outside of Gygi lab, Rosa Viner, and Simion Kreimer -- I'm not sure anyone else ever used it. Why?

1) The experiment was nearly impossible to set up.
2) The data was nearly impossible to process.

What's on my desktop? Right now? 400 icons and a picture of Isaiah TomCat sitting on the head of a pug...AND...

.....A WAY TO EASILY CREATE TOMAHAQ EXPERIMENTS AND PROCESS THE DATA!  Which is the reason I'm writing this! 

Multiplexed internal standard triggered multiplexed targeted proteomics. For everyone. You can get the TOMAHAQ Companion software here. If you're paywalled for the paper, there is enough information within the GitHub download to set everything up.

OpenTrons OT-2 in the house!

YEAAAAAAAH!  Finally here! More details will certainly follow as we (well... @jenkins_conor) writes the software to make this thing:

1) Do BCA assays (whatever those are)
2) Digest things
3) Phosphopeptide enrich things
4) Extract metabolites
5) Dilute extracted metabolites

So far I'm impressed with the thought that went into the setup, the quality of the materials, and Conor said the developer software for it exceeds expectations. The fact it was 1/10 the price of any other one I wanted didn't hurt either...

You don't get a PC with it, but it connects via WiFi and you may need to have some Python guru hanging around to make it do new things it doesn't know how to do. Of course, method development and validation will take some time, but the manufacturer hosts methods developed and posted by other labs. We'll obviously upload ours as we develop them, if the robot is any good.

We're currently using an older Chromebook that is now running Linux, but the thought of getting a Linux tablet for it once we're confident of the performance and reproducibility and sticking it to it with a magnet is seeming like a solid idea.

Nonhuman proteins in human milk?!?!??

(Image is from @Confused_Cow, a very strange Twitter account I am now definitely following)

What I actually want to put here is this confusing new JPR paper....

Yeah....okay...so....ignoring the biological implications that sound like this would be completely and totally biologically impossible (all the proteins we eat get chopped up by that stomach acid stuff, right?), let's look at the amount of work that went into this ---

6 humans donated milk.
This milk was separated into 6 fractions by SDS-PAGE
These SDS-PAGE fractions were analyzed using a passive split flow Agilent nanoLC system in what looks like around 45 minute gradients on a Q Exactive Plus system
MS1 resolution of 35,000
Top10 experiment
MS/MS resolution of 17,500 with a 120ms fill time.

The data processing was done this way --

--I've highlighted the things that I find interesting here, and that I would have done differently.

Wooooooo.....okay.... In one sentence -- This is a really big search matrix searched against masses obtained at medium level resolution, using search tolerances that are strikingly high and searched against a software package version that has been lampooned for it's inability to accurately quantify FDR when matrices get really complex. Yo. If you're Q Exactive Plus is off by 48ppm, get a fire extinguisher. No QE+ that isn't currently on fire or under water is off by that much.

EDIT/Clarification: Proteome Discoverer 1.4 only controls FDR at the PSM level. All later versions of PD were designed to control FDR at multiple levels in order to more accurately control data quality with extremely large numbers of spectra or with high complexity search space.

Sorry, but this seems like an invitation to a list of proteins that are gonna be bad.

HOWEVER.

If you do your searching of your global and then validate the results with Parallel Reaction Monitoring (PRM) -- and it works -- maybe I should shut up.

Particularly, I guess, if you make heavy isotopic standards as well for your PRMs and this stuff looks legit. And if you go to the supplementary info -- it looks on point --

-I should probably definitely shut up.

In the end -- I'm still confused. WHY ARE THERE DOG PEPTIDES IN HUMAN MILK?!?!?

But the work looks solid.....