Wednesday, December 12, 2018

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.

Tuesday, December 11, 2018

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.... 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.


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.....

Monday, December 10, 2018

Assessing automatic sample prep technologies!

Was this already on here? I forget. And does it really matter anyway? This is cool enough to talk about more than once!

Honestly, in general, all these robots are freaking me out a little. It seems like every time I go to a store or leave a parking lot, there is one more robot thing and one less person working. And it's weird.

However -- in science, where reproducibility is kind of insanely of completely paramount somewhat importance -- automation is amazing!

Max Planck is using the Agilent thing.
This new study used a bunch of things -- and the reproducibility is kind of insanely completely awesome looking -- even on samples that are prepped on separate days!

Sunday, December 9, 2018

OMNITRAP -- Upgrade your QE to do EVERYTHING!!


Apparently, I've been under a rock for a while. Or hiding from my...

...few unanswered emails.... Don't worry. It is mostly because I bought a compost bin from Amazon in 2015 and now obviously I'm a compost bin collector now.

I'm driving home from horrendous holiday shopping craziness and my wife is flipping through the new Science and less cool Scientist and reads off something about Roman Zubarev (Ben is paying attention) and then reads off details about the "omnitrap" (Ben is having trouble keeping a hybrid in it's lane) and then "that it's an upgrade for any Q Exactive! (Ben requires moving from driving to being the passenger).

You add the OmniTrap to the back end of your Q Exactive.

It costs you about $250-$300k

It gives that Q Exactive some new capabilities. Like --

I don't even know what all those things are!  I just know that I don't have them. And I sure would like to.....

Saturday, December 8, 2018

Split resolution control on the QE HF Turbo!

Okay -- am I an idiot? Sure. Yes. Totally. But after a night of broken Orbi XL and-still-not-working after the rainstorm Orbi Elite AND the HF slEasyNano on the fritz again -- maybe being able to FINALLY GET AN MS1 RESOLUTION BETWEEN 60,000 and 120,000 is cause for celebration!


Mark Ivanov and
Elizaveta Solovyva

of the great Gorshov lab for taking a look at my data, finding dumb mistakes that I personally made and showing me awesome new tools I can't talk about quite yet.

To be clear, they weren't trying to help me replicate Jenny's phone number on my Orbitrap. They were being really brilliant and helpful and, as a consequence, I did something stupid while verifying I finally got the stupid nanoLC running again. But -- nowI can run my samples at a resolution right in the goldylocks zone. 60,000 just isn't enough for digging deep and 120,000 takes too long.

86,753 MS1 resolutoin
15,009 MS/MS. YEAH!


Friday, December 7, 2018

MaxQuant.LIVE Workshop in Zurich next week!

I know the first thing you probably thought when you read that subject line. Same thing I thought --"what's a Zurich". I'm happy to tell you that it's a city. And either something is very wrong with my Google search preferences OR the most important image to tell Americans about Zurich is that Rihanna has been there.

Now there is a second reason to go wherever that is!

Is this the world's first ever MaxQuant.Live workshop? I think it might be!

I love ProteomeXchange. Don't download this amazing dataset yet!

Okay --- so how ridiculously cool does this sound...?

Did I download this dataset? 

Will I consider sending grumpy letters to a bunch of journals if I'm not able to read about this ridiculously cool study STAT?

Are these questions rhetorical?

Oh -- and on that topic. This is one really interesting page of text from John Yates, III. that you should check out if you haven't seen it already. 

I think it spun off a lot of Twitter conversations that I need to catch up on from my metabolomics hiatus.

Thursday, December 6, 2018

Plasma profiling -- Real patients -- post gastric bypass surgery!!

I'm going to geek out over every detail of this one later. Unfortunately, in a huge rush right now.  Honestly, I may spare you from it. I'll save it for trying to convince people in my geographic area that mass spectrometry is more than just an experimental toy.

Punch lines?

175 patient PLASMA proteomes

>1,000 proteins when each was ran in triplicate (no depletion)

This goes down as THE largest quantitative dataset on non depleted real patient plasma (prove to me that SomaScan is quantitative and I'll consider changing my mind on this statement -- and comparing your array to GWAS doesn't count, yo.)

45 minute runs on an HF

Automated the whole sample prep thing with a robot. Heeeellllooooo fully functional clinic-ready assay!

Umm...not what I was looking for...but too funny to leave out.

Wednesday, December 5, 2018

A comprehensive pipeline for translational top down proteomics!??!?

Okay -- so -- I can't read this yet. But I can see the title and it sounds impossible.

"Translational" is the word we use when we mean "WAIT. WHAT?!? WHAT I'M DOING RIGHT NOW  isn't just a curiosity -- this could actually help someone!!", right?

"Translational top-down proteomics" immediately made me think of this.

But....we've seen some stuff recently where the practicality of applying top-down to serious far-reaching problems has actually seemed realistic. And -- I've gotten close to 100 digested samples across my Fusion in the last 3-4 months on this protein Ntai et al., conquered unequivocally with top-down -- and I think digesting makes it almost impossible to work with. Take a screen capture before I edit this: I think one of the most important proteins in cancer can't be studied effectively with shotgun proteomics.

It still seems like a long shot for helping patients. Most hospitals still haven't embraced the power of them new-fangled triple quad things. But maybe not. And we won't know till we try, right?

Tuesday, December 4, 2018

MS/MS visualization in Python!

I do love this python mass spectrometry community. Keep it coming, y'all! This is simple, the output is nice, the code is open...everything you could want...