Shiny + MetaProteomics + gut microbiome = pepFunk!

Did I spend more time looking at "Funky" gifs than using this great online ShinyApp?

(Funky BigFoot seems to...imply...yes...?...)

Inspirational paper? This great new tool!

Wow -- the the Gifs are making me a little dizzy, to be honest.

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(.....toooo pepFunkyTown....)

Despite my clear focus in this blog post, the "pep" in "pepFunk" is the central story in the paper. These authors find that when doing gut microbiome metaproteomics (i.e. digesting poo and trying to figure out what is there) that the peptide centric approach is a much better indicator than assembling to the protein level and then trying to interpret.

That makes a ton of sense because the whole technique of rolling up incomplete data at the protein coverage level to incomplete data at the FASTA level (since we don't have a true database of every organism that is present or their distribution) makes even less sense in a population this diverse than usual.

This isn't a suprise, but how do you utilize peptide centric data? Not easy -- until you've got a simple web app to dump data into!

You can check out the online version of the pepFunk Shiny App here.

Mixed Data Acquisition -- DIA + DDA in the same run!

Which is better?

Data Dependent Acquisition (DDA)
or
Data INdependent Acquisition (DIA)

Presenting -- MIXED Data Acquisition!

To help clarify this idea, I outsourced the design of a schematic of a "tribrid" orbital mass spectrometer device to some professional illustrators.

Tricked you! I did...part of this...myself! 

When you're doing DIA in your fancy instrument, the ion trap is doing a lot of nothing. Why not acquire DDA MS/MS spectra there while your Orbitrap is busy getting MS1 and DIA data? It's like spectra for free! 

Obviously, you'll need to worry about cycle time -- which is tough to calculate on your own on the Tribrids -- and consider the matrix and chromatography time, etc., etc., but there are key advantages. 

You know those coeluting peaks that kind of smear together in the XIC, particularly in the high mass range where your resolution is lower? These authors demonstrate that they can use the DIA windows to separate those out and improve their quan. The paper is pretty short -- proof of concept, but it doesn't take much imagination to see the potential advantages. Use your MS/MS to generate your libraries that you use for DIA quan? 

If there is a downside, I sure don't see it. You paid extra to get that ion trap back there. You might as well make it do stuff!  

HLA Peptide Sample Prep Completely Changes Peptide Populations.

This is a thorough analysis of HLA peptide enrichment and purification strategies that finds -- wait for it -- that how you prep these awful terrible poorly fragmenting (highly deamidated?) and often modified peptides changes the ones you're able to see.

I think they did a great job for this work, so the following should not -- in any way -- be considered a reflection of what I think of the work this team did.

From the abstract --

Again -- our biggest weakness in any head to head with any moron with a Hi-SeQ (or who has $1k to spend on a NanoPore -- which -- is coming fast) is that proteomics, as a field, can not ever once follow the same sample prep procedure, is completely and utterly, in every way, our fault. The moron with the Hi-SeQ just has to follow the directions that came in the box.

Proteomics Data Mining Challenge (#ALSMinePTMs) Data Coming in!

Good morning/afternoon/something crazy world out there!

Tons of data is coming in for the 2020 Proteomics Data Mining Challenge, as well as questions/clarifications (RIP my Inbox)....

If you've been expecting an email from me, it might possibly be in the 4...2...8...?...drafts...?

I'll investigate that...later....or just delete them. That seems unconquerable.

Just a reminder that I am not a judge, so if you attempt something like the submission at the very top -- I'll 100% appreciate it, but it won't help you out. I'm just the hype and disorganization guy.

This is a lot of data and if you're running a little behind, just let us know. I'll try to get to the questions that have come in today (...sorry...)

We're getting data via WeTransfer, DropBox, BOX, GoogleDrive, and something that started with a V or a Z? Meh. Seems to have worked, as well as data in CSV form that has been small enough to email.

WOOO!! Let's go!

London Proteomics Meeting on SARS-CoV-2 this Friday, May 1st!

Not tired of SARS-CoV-2 yet?

Maybe you need to hear someone with a squeaky nasal voice talk about it in a West Virginian accent.

If so, you're in luck!  Somehow I've been added to a lineup this Friday for the London Proteomics Discussion Group.

Two qualified scientists will also be speaking!

You can register for it here.

JPT is killing it on the SARS-CoV-2 standards!

JPT is one of those companies that I think everyone forgets about, because they've been around long enough and made enough great standards that everyone has heard of them at some point. I, for one, can never remember the name and it helps if I start thinking about BASS CANNONS. (Changed my mind -- an Excision link does belong here. Used to tour as the loudest act on earth. Not kidding.)

If you don't know about JPT -- they make my favorite retention time standard for proteomics, PROCAL. Which not only has super sensitive chromatography metrics thanks to the really clever way that the peptides were designed -- but it also can be used to match your collision energy from instrument to instrument!

You can tell the labs that use it because they'll have multiple instruments in their method files and the Tribrid is at 32.5 HCD and the benchtop is at 26, because they used science and standard to match them!

Now -- I'm not just beaming about JPT solutions because I'm pretty sure I keep forgetting to pay them for HLA peptide standards I bought this spring (which they've never once reminded me about -- and are the only place I ever order them from, because they are suuuuuper clean).

They've been killing it on the dumb virus thing we keep talking about. You can basically buy peptides covering the entire virus -- and now you can buy convenient sets!

Metabolic Precision Labeling of O-GalNaC glycosylation!

Glycoproteomics is rapidly massively evolving right now, everyone. It seems like every week some reason that makes glycoproteomics an awful and terrible thing to do with your life is solved -- or, at least, mitigated...

Next addition? Selective labeling of O-GalNac! 

Sound boring?

It is until you remember the best part about glycans in mass spectrometry! All the stupid sugars have the same stupid masses!

Is it a serine GlcNac? Or a serine GalNac? I dunno. It has the same exact stupid mass. If you really try with optimizing the fragmentation, the distribution between the fragment ions will look a little different. Fortunately, which one it is doesn't matter to biological systems!

Living systems can be annoyingly particular about what sugar isomer is where! And with this system, I don't totally understand, you can selectively label O-GalNacs and tell them apart, not only with mass spectrometry, but also with microscopy!

I don't have to understand the labeling methodology to get that this is a potential game changer that can light up big questions in biology!  I'm so confident of that fact that I only skimmed that part of the paper.

SimpliFi! Simple, powerful proteomics data interpretation software!

To start -- SimpliFi is a commercial product and is the property of Protifi. I can't make trademark signs, but both those words have those.

Next, however, this is critically important. Ingenuity Pathways Analysis (also gets a TM) is crazy expensive, and was never ever meant for proteomics data. It shows, and I think it always will. But what else do you do? Holy cow, maybe you SimpliFi!

I saw a demo of this at ASMS last year, but it was an outline. If you caught the day 1 virtual US HUPO talks (which you still can here!) John Wilson gave a talk on this program -- and he undersold it. I've been messing around with a demo of it, and it's a game changer for 2 main reasons

1) It make you think about experimental design. (Something proteomics, as a field, isn't real good at)
2) It puts a lot of power at your fingertips in an intuitive way (except for the exerimental design part)
3) The output is proteomics centric (who makes a list with 2 things?)

You should just ignore me and try out SimpliFi here!

I registered and got a demo code to try it out and the data import is fantastic.

You upload your CSV results from whatever you want. SimpliFi then makes solid guesses about that data you gave it. If it is wrong, simply highlight the columns or rows that are correct.

Have you uploaded data into ShinyApps or into Ingenuity? How much time do you spend going back and reformatting your stupid CSV file so that it will identify your data correctly? 99% of the time you've blocked off for your data interpretation? Oh -- is there an invisible space in your CSV? And is that why the browser window locked up for 3 hours while you thought it was processing?

Not a problem here!

Also -- and I'm scared to hope -- for real -- but SimpliFi allows you to put your data in with the distinct batches your files were in. We're thinking about BATCHES -- IN PROTEOMICS?

(Not the game?)

More on that later when I dig up some data where I can test it, right now I'm messing around with some cool cancer demo data -- and look how beautiful this output is!

Okay -- yes -- if you're an expert with R and or Perseus, maybe you won't find this as impressive as I do, but if you're not an expert in this stuff and you're constantly flabbergasted by requests to interpret the beautiful data you just generated -- this may be what you're looking for.

I think it's still in demo/development phase, and we might find out it costs as much as Ingenuity to use when it comes out, but in just one day of messing around with it with classic NBA games playing in the background here and there (Iverson is under appreciated, btw, Hall of Fame or not.) -- I'd write a justification to trade the two out right now.

SARS-CoV-2 PRM Assays! How low can we go?!? As low as the other diagnostic assays!

This is big!

In our preprints we proposed what it would take to make LCMS a competitive diagnostic assay for the other SARS-CoV-2 assays out there. We know we probably can't beat RT-PCR, but we need to hit a magic number the ImmunoSwab assays and ELISA's and Protein Arrays can hit, because they've been validated to work!

That magic number is around a detection of 20 picograms viral protein/mL biological fluid.

And these people just showed you can hit that with PRM! 

They used an Orbitrap Eclipse.
They used 60 or 90 minute gradients.
They used NanoLC.
You could argue none of these are ideal for clinical diagnostics (unless you're the manufacturer of the Orbitrap Eclipse, which, would probably argue that's a great idea at a little north of $1M USD per box), but as a proof of concept?!?! This is fantastic.

The files are at ProteomeXchange/PRIDE here.

Females in Mass Spectrometry -- register for the next meeting April 30th!

Important details --

More importantly, all the cool people are doing it!