....Appropriate music for today....

Logging into ASMS Reboot!

Confused about how you get into ASMS Reboot?

Me too, but Dr. Ashwood has us covered!

Wait. Okay. I think if you click on the Tweet image above it will take you directly there. Wait. I'll make a button!

HIT THE BUTTON BELOW TO GO TO THE ASMS 2020 REBOOT!

Great COVID-19 webinar and Mass Spec diagnostic clinical trials underway in Europe!!

My quick overview

#1 MALDI-TOF with a low(er) resolution bench top instrument -- maybe 90 minutes of sample prep to inactivate the virus and spot gets you to 3 minutes on per spot.

Beyond COVID-19 -- Dr. Iles made it clear that what they're doing with this stupid virus would work really well for other viruses!


#2 -- is the paper that Wiley Proteomics accidentally paywalled and I was a jerk about (they fixed it)

Actual paper link here!

I'm going to mix up what I'm taking from the second talk in the YouTube thing above and from the actual paper, but I'm too excited to try getting it straight.

Which is a really nice study that shows 20min DIA and 8 min LCMS (QQQ) using acetone precipitation and 4 hour tryptic digestion.

Correlation with the gold standard WHO approved qPCR assay? r(squared) = 0.887 !!!

They can get to 180 samples/day and point out that in Ghent alone, they have 20 comparable instruments that could be used to help relieve testing backlogs.

Okay -- THEN -- they bring in 80 people to help! And they optimize, optimize, optimize and spike in QconCAT as internal standards and then even push the data processing with machine learning -- sped up the digest (to 15 minutes?!?!?) and push the accuracy even higher.

That great talk ends with this beautiful and inspirational slide -- maybe -- just maybe -- we should stop making fun of how old and slow PCR is and how stupid antibodies are and work together to get to the number of tests that we need to?

....I'll...try....

COVID-19 MS Coalition -- And tomorrow's London's Proteomics Discussion Group (5/29/2020)

I know I mentioned this cool thing before -- but there is also a paper now!

Hell yes. If anything good has come out of this global pandemic it has been highlighting how underpowered traditional diagnostics -- and clinical medicine truly is. Time to kick some insurance companies and hospital administrators to the side and let accurate instruments like mass specs start filling bench spaces (once we confront some of our remaining demons). What better way to do it than with tons of people working on it?

Also -- tomorrow is 2 more great talks on COVID-19 proteomics from the London Proteomics Discussion Group.

Tomorrow we'll finally see the BioTyper-type MALDI-TOF analysis of SARS-CoV-2 infected materials that we've been hearing about in the media. And we get to see more targeted peptide diagnostics by LC-MRM!

You can register here!  And then get your rest, cause it's ASMS for real!!!

Wiley wins! We knew someone would paywall a COVID-19 paper!

EDIT -- It was just a mistake. I'm an impatient jerk. You can read the paper at the link below and read my interpretation of this great work here. 

Go Wiley Go! Get that money! 

I'm sure this is an honest mistake, and I'm just drawing attention to it so they can fix it and I can read this paper. 

Edit -- 944PM EST -- it's fixed! I'll put a real note about the paper in here when I have time. 

Holy shit. Is it ASMS already?!?!?

Monday is June 1st!  Time to start our favorite impossibly exhausting week of the entire year -- sorta... Things are already starting....LIKE...?

Skyline User Meeting was today (5/27) and tomorrow (5/28)!!

You can see what happened today or see the great talks tomorrow by going here (register for tomorrow -- we'll see MSAmanda in Skyline and other cool stuff).

From today's stuff (which was totally great, except for my 5 minutes of not understanding how Windows 10 works still....how can it be this bad...?..where are my thumbnails....?) -- my recommendation:

If you're a core lab -- definitely check out Roman Sakson's "Skyline in core environment" -- what he does with locked down layouts for sharing data is stuff I've never seen before and I hope other people will start doing all the time.

Lipids and ion mobility and hunting clinical biomarkers round out the first day. If that's your jam, it's there.

Big shoutout to whoever just typed "Go Hokies" -- I saw it before the moderators dismissed it.

User meetings for vendors are in progress or start tomorrow!?!?!

Okay -- so -- I need more time to digest all this -- BUT THERE IS A BUNCH OF NEW HARDWARE!!

There are 2 new Exploris instruments (the 120 and the 240)

Mandatory --

The best I can tell, the 240 is an Exploris 480 with very few limitations (max resolution 240k) and a new paint job.

AND -- ACQUIREX added!   You can check it out here.

The 120 appears to be an Exploris focused on small molecules. I'll read more later, but it's up on this here website.

Something to pay a lot of attention to? The world's 3rd worst acronym -- uHTPPP -- Say it 3 times fast -- and then -- consider what Ultra High Throughput Plasma Proteome Profiling might do for you. And then say it 3 times fast again. Outloud, please.

Other vendors have things as well. I'm really interested in this Acoustic Ejection thing. I think SCIEX talked about it last year, but you couldn't actually buy it yet.  The QTOF still isn't ready, but the Triple Quad is. I've got some budgetary numbers and it's about the same price as a stripped down Orbitrap Eclipse -- but...can your triple quad run 2,000 samples today?  Its out of my price range, obviously, but if the sensitivity specs aren't made up....it's massively impressive. There is an extremely vague video and a clock that is ticking down to something? here!

For less vague information, check out this preprint from January.

Bruker isn't just sitting around, but it's a lot more applications focused this year for them. You can see a lineup of their user meeting talks here.

Talks I will try not to miss?

P.....R.....M.....at 100Hz(!?!?!?!?!?!??!)
ZipChip integration! Okay -- this big dumb thing is fast enough for what I keep trying to use a ZipChip for! Fingers crossed.

Revisiting gas phase fractionation with today's technology!

Every few years someone (actually -- pretty commonly, one guy in Wisconsin) gets fed up with HPLC entirely and tries to do something smart so the pumps can finally be dropped off the roofs of the parking garages.

It always seems like the technology is just....almost....ready....

Here is one I found particularly promising 7 years ago....

We've come a long way since then! Are we there yet?  Maybe? Check this out! 

132 samples in 4.4 hours? Okaaaaaaay -- that's something.....

The name is...meh...for a method out of Wisconsin. Don't worry, Twitter, has it covered --

--what about.....

Meta-Analysis of 1,000 Proteomics Experiments in the Search for Missing Proteins!

This is a really interesting study describing the progress toward finding the "missing" human proteins (the ones that genomics says should be there, but we haven't been able to find protein data for).

--and reanalyzing a few (1,000!) of the files toward just one chromosome of the Chromosomal Centric Human Proteome Project (C-HPP).

An interesting take-away for me from this study is that it appears that we are approaching a state of diminishing returns due to how much effort goes into the identification of so few peptides. We've found the easy ones, I guess! Time to think up some new strategies?

Rereading -- FAST and Low-cost detection of SARS-CoV-2 peptides by LCMS!

After a few hours of sleep, let's looks at this awesome new preprint again!

What's it do? Well...it tries to establish our beloved LCMS systems and shotgun proteomics work into a real life diagnostic assay for the virus.

How'd they do it? They got some infected nasopharyngeal swabs and removed 200uL of the material for digestion.

They used SP3 digestion overnight (following reduction/alkylation) they prepped with a Hamilton Starlet --  I think it was this one, but this might be a newer model?

They used microflow at first on a QE HF-X. And that was fast -- that was like 9 minutes.

Then when everything looked great -- they went Turbulent Flow(??) with a Triple Quad (TSQ Altis) and cranked up the speed dramatically.

Do you know about these multiplex HPLC things? Here is an example image.

You see them in biopharma labs all the time. You run multiple gradients simultaneously and you either do the UV Absorbance or mass spectrometry on the point in each gradient that you're interested in.

For example, if your peptide of interest comes off at 33 minutes and that's all you care about, why waste that precious mass spectrometer looking at the whole gradient? Just switch over at 32 minutes, ionize till 34 and switch to the next column and gradient. HPLC pumps are $30k? Switching valves are $2k? 4 pumps and 2 switching valves and you're doing the work of 4 LCMS systems for less than the price of two.

Now...your plumbing flowchart kinda looks like this in the end ---

-- but as long as nothing goes wrong, you're getting high throughput on the cheap!

I'm not familiar with the difference between high speed multiplexing and Turbulent Flow, but I think this all still makes sense.

This is how they get to crazy throughput. In theory -- 500 individual samples screened per instrument per day. Which is fantastic!

Again. Great paper. I should sleep more. And read slower. Awesome work and a huge congratulations to this team.

It's glycoproteomics week! SugarQuant, MS-Fragger-Glyco and O-Pair search!

As a silver lining for this virus thing is that we've got lots of time to work on informatics pipelines! How much? What about enough to drop 2 new glycoproteomics workflows the same day?

The first is an extension of the ultra fast MSFragger workflow and the figure at the top does a great job of explaining what it is doing. 

The second (and in no particular order, when I select multiple images, blogger inserts them however it feels like) -- appears to be a new addition into MetaMorpheus, called O-PAIR search! 

I unabashedly love both of these software packages, but don't have the time to test them and write glowing things quite yet. 

On the glycoproteomics side of things I also have to point out this thing! 

SugarQuant is more than a software package -- it's a new twist on just doing glycoproteomics in general. TMT labeling + SPS MS3 for glycoproteomics and it utilizes ALL THE DATA. 

The software (GlycoBinder) can be found at this Github!

Whoa! Some guys came out of nowhere and got $100M to do proteomics!

Man....I've got the wrong friends....

Okay, I get it. The ProteoWizard guy does have a bunch of citations, but....ummm....almost $100M to do some vague proteomics stuff with some guy who definitely has not background in our field?
Good for them! If this turns into some silly array stuff, I'm going to vomit.

Check these articles out!

This one is even more interesting. Stealth mode proteomics.

I assume with all that money they'll be hiring a bunch of great people out there to do whatever it is that they are doing.

Amateur hour is over -- Mass Spectrometry Diagnostics for COVID-19!

EDIT: Better read through of this paper posted here.

When it is time to make a new clinical diagnostic. The ONLY time that mass spectrometry is considered.

The only time.

Is when there is literally no other option. No matter how bad that other option is.

People will bleed every drop of blood from every rabbit on earth before looking toward the most accurate analytical instrument ever developed on earth.

Well -- my friends -- in COVID-19 diagnostics, we've literally tried everything and we're out of options.  I guess it's time to go to mass spectrometry!

Wanna check it out? Or do you want to go suck the blood out of some rabbit and try and convince me that whatever that rabbit blood does is reproducible? If it's the former, here is the link!

They do optimization with the QE HF-X
They do some ultrafast targeted assays.

They find that if they've got 9 min per run they can kick out 84% accuracy in diagnostics.

And here is the fun part! If it's negative by PCR but positve by LCMS who are you going to believe?

Something that heats up fragile nucleotide strings and stuffs things between the strands, cools down and repeats?

OR

Something that measures a fundamental constant in the fucking universe (mass to charge ratio) directly. I dunno. I guess I'll chance that PCR thingamajing? Who doesn't love an indirect measurement?

Or something that says --
Negative (noise!)

And

Positive!! (Peak!)

Look, I know this isn't going to get out of my little bubble of mass spectrometrists that I preach to all the time, but COME ON.

People are doing spectrophotometry on colorimetric assay reagenst right this second in hospitals across the country to crudely measure one protein!

And here we are with cool research tools that can do so so so much more....

Maybe it's time for us to step up and throw slow, annoying, insensitive and inaccurate diagnostic assays to the side and just take over!!

Phosphoproteomics of SARS-CoV-2 infection suggests new treatment options!!

I'm guessing you've probably seen it, but if you haven't, you should stop and read this.

As a field, we've been messing around with phosphopeptide enrichments at least 12? 15 years? And -- yeah -- it's cool that we can pull down phosphorylated peptides and identify/quantify a bunch of them, and put them into big databases. Sometimes, though, it feels like we're just going through the research motions.

This is an actual real-life application of phosphoproteomics that shows -- it's actually useful. Not to put down anybody else or the thousands of publicly deposited studies, but this group just used our very standard research experiments to find new ways that we could potentially inhibit the replication of a virus that is fully confirmed to have killed 1,425 people in my country yesterday.

HOLY COW. Wait. Can proteomics be helpful to the world? Is this one of the tiny silver linings in this global catastrophe?

This blog is supposed to be more on the technical levels of how to do this stuff. Or just the ramblings of some guy who reads too many scientific papers while experimenting with the LD50 of espresso.

How'd they do it?
SP3 based digestion
TMT10-plex labeling of 125ug of peptides per channel (to 1.25mg total peptide)
250ug of the total peptide amount was fractionated (high pH reversed phase with a big (4.6mm Waters Xbridge column -- [I think the same one that Jesper Olssen's lab uses for the high resolution fractionations, but don't quote me -- I always use the 2.1 mm one, but I may need to scale up, both group's data might be better than mine...but both have better instruments....worth investigating....]) into 96 fractions over 70 minutes
The 96 fractions were concatenated to 24 for LCMS

The remaining 1mg of peptide was enriched with Fe-NTA and then fractionated differently. They used C-18 stage tips, fractionated to 8 and then concatenated to 4.

Two different LCMS methods were used on the Fusion Lumos --- SPS MS3 for the whole proteomics (Ion Trap on Turbo, MS3 at 50k resolution)

MS2 based quan at 50k resolution was used for the 4 phosphoproteomics files.

At first I was a little thrown, but the real reason that SPS MS3 is useful is coislation interference and ratio suppression. When you do a relatively simple mixture, I get a lot less improvement from MS3 over MS2.

Also -- it's phosphoproteomics. Do biological systems function with a 1.8-fold upregulatation of a kinase changing anything? I dunno, but it sounds unlikely. When I see a real phosphorylation event it's typically 5-fold minimum. A lot of the time it's a huge difference.

PD 2.4 for data processing. I'm a little lost on how the data was combined with ZScores, but Siri is telling me that I have work to do, so I'm gonna wrap this up.

So.....the phosphoproteomics points them to some things that they could inhibit to reduce the viral replication -- so....they

INHIBIT THEM

AND TEST IT

AND IT WORKS

Offset multiplexed targeting -- The brilliant targeted method you should have thought of first!

I have been dying to talk about this paper and it is finally out!

If you have been running a mass spectrometer of any kind -- get ready to feel reeeeeeaaaaaallly dumb that you didn't think of this first.

I'm allowed to say that I was lucky enough to be one of the reviewers if this, right? And this extra time has been very useful becaus my ego has almost recovered from never once thinking of this myself.

If your name isn't on that list of authors above, I'm going to now refer to you (and myself, of course, because I'm just as guilty as you are) affectionately as...I dunno...let's go with "Dumbass"

Hey Dumbass! You know how you can get a heavy labeled peptide for anything you want? Heck, you can even order complete heavy labeled peptide mixtures?

Oh -- you knew that? Good. So did I.

Hey Dumbass! Did you know that you can offset your fragmentation window?

Oh -- you knew that too? Yeah. So did I.

So....Dumbass....did you ever put these things together and think -- I could just put in my heavy labeled peptides and when those triggered, just offset my fragmentation backward to where the light peptides would be?

No? Neither did I.

I can't overemphasize how much I am going to use this technique. You can essentially bypass all the challenges of PRM with the addition of a heavy peptide standard that you target and then offset.

You need to do a little bit of math to work out your offset, if -- for example -- I just wanted to characterize a pathway of interest, all you need to do is get heavy peptide standards for that whole pathway. They make kits for these.

There are complete whole protein and phosphoprotein heavy peptide standards for entire pathways....

...as well as for critically important central proteins like TP53 which are almost impossible to monitor with traditional proteomics due to their exceptionally low expression levels! 

#ALSMinePTMs final data submission deadline this week!

We've had some great submissions for the #ALSMinePTMs Proteomics Data Mineathon Challenge and we're about to get them summed up and over to the judges.

If you haven't sent your submissions over because your life has been disrupted by some pandemic thing, we'll still work you in this week. If you're aaaaaalmost there, shoot an email over and we'll do what we can.

Submissions can be sent by DropBox, GoogleDrive, BOX or even summed up in a powerpoint, Excel sent to:

lcmsmethods@gmail.com

If I had to choose one webinar to attend this month? Stats in Proteomics!

Who spent 11 hours on phone calls and Zoom webinars yesterday?

Everybody!! 

Okay, if you're getting a little fatigued with all this amazing remote learning and need to be a little more selective about what you register for -- what's a big huge problem with proteomics that we can fix from home by designing our experiments a little more smarterishly? And analyzing them with some more robusterlificness?

Experimental design!

And next Tuesday there is a talk at noon courtesy of this cool organization that you can grab a salad (lunch where I'm at, and I assume that people in California eat salads for breakfast) and get a statistician's perspective on how to make your proteomics better.

This is free and you can register here.

MCP is going full open access!!!

As another great step forward -- MCP just announced, along with whatever ASBMB is (for some reason sounding it out reminds me of a Denzel Washington movie, not sure why....) that in January 2021 they're going full open access! 

Will the editors and reviewers still be some of the hardest people to impress in all of science? 

Of course!  (It's still MCP)  

What it does is opens a very important door. 

1) For the journal to get more real life exposure for the nitpicky, professionals-only, crazy rigorous, stuff that it will accept. 

2) And for it to be easier for the outside world to see what we really can do in proteomics when we absolutely have to stick to rigorous standards for study design, data formatting, and interpretation, etc., etc.,. 

Look, I know impact factors are stupid, but open access always improves how much your study is read and cited (this study found an 18% increase) and I suspect this will be a far larger increase for MCP.  How many of us really write when we're at work? I think the number is lower than other sciences. When we're in lab there is a nanoLC that requires....something....they always require something.... or there is a biologist that needs to talk about data interpretation of a disease they're supposedly an expert in, plus the vacuum pumps are SO LOUD. 

I think most of us write in the early mornings or late at night and if I've got the choice of logging in through VPN to my library or using an open access reference that will just open through Google Scholar? I dunno about you, but that's almost always an easy choice. 

London Proteomics Discussion Group (More SARS-CoV-2) Friday 5/15!

This Friday (May 15th) there is another of the biweekly (bimonthly?) -- every two weeks -- SARS-CoV-2 proteomics talks. This Friday's will feature --

You can register here!

The Clinical Knowledge Graph -- Is this the missing downstream piece!??!?

This deserves FAR more time than I have right now to give any one single thing right this second, but if it is 1/10 what I think I'm looking at, it's amazing and you should check it out.

How bummed out do you get when you give someone a perfect dataset -- you know the one, the one where the QA runs before and after were perfect. The one where you didn't need to impute anything. The one where you didn't even need to apply normalization because your loading was spot on in every injection. You hand that over knowing that at a technical level you've done EVERYTHING as well as anyone in the world could do it.....

....and you get the blank stare from the collaborator.....and that "what do I do with this?"

And what do you do?
Your first thought is to say something like, "Wait. What? Aren't you the f****** biologist? Isn't this the disease you study? Go do f****** biology with it... don't you dare tell me you brought this here with no plan of what to do next!?! What do you actually do here?!? F*** right off and get out of my office, F***!"

But that isn't what you can say, because you weren't raised in West Virginia and you were taught better manners than that. Instead you point them to the 4 resources that probably make the most sense for their study of the 50 or so that you know probably could be used to help them, because -- well -- we really don't have centralized proteomics knowledge or even protein level data. It's spread everywhere.

Maybe the Clinical Knowledge Graph is a step toward fixing this. Maybe? I can hope until I can put some data into it!

The Metabolomics Spectrum Resolver -- Visualize multiple matches!

A super fun exercise if you're new to metabolomics is to go ahead and search your experimental data against the wrong database. Working on human metabolomics? Try using a library for pesticides or archaea or from the fossil fuel industry or something. If you're using MS1 for identifications, your number of IDs may not even decrease, particularly if you're using resolution less than 30,000! And due to the similarity of many small molecules you'll find a lot of positive hits with MS/MS as well.

In a more realistic experiment, there are MS/MS libraries for small molecules all over the place. If you get multiple conflicting hits for the same molecule from different databases, what do you do? (It's common to search your data against multiple libraries -- and multiple conflicting matches is extremely likely). Interpreting which one is correct is often a nightmare because there is no format unifier.

BOOM!

This online interface can directly import spectra from 6 different metabolomics databases and compare them. The format is fantastic -- it looks similar like the IPSA program from Coon lab for annotating peptides -- it is intuitive and the graphs are pretty and can be directly exported in a several different formats, from Support Vector Graphics through JSONs and less weird stuff in-between.

FDA/NIH Glycoscience Day is Full Remote!

Every year I think -- I should put in another abstract for the NIH/FDA Glycoscience research day.

And then I remember how much fun it is to drive to the NIH Bethesda campus during rush hours.

I'd sign up for this instead EVERY. SINGLE. TIME.  

Honestly, where do you guys use the bathroom when you're it takes you 45 minutes to move forward 3 exits?  Is "bladder of steel" listed in the job requirements down there? 

As something positive to come out of this virus thing -- the whole thing is remote! 

And it isn't a bunch of local government shmucks talking. They got Rebekah Gundry who will talk SurfaceGenie and some other heavy hitters from the real world in the massively growing field of glycomics and glycoproteomics!  

You can register here!

A new crisis -- A shortage of peer reviewers!

Amidst the crises of this virus thing, we've got another one going on. No one can seem to find peer reviewers. As much as I make fun of the speed and other weaknesses in the peer review process, it is obviously still critical to scientific progress.

We've got two papers out where the Editors have told us -- sorry -- no one will review this. Now, since my name is on it, you could just assume that they're both really really bad, or I've successfully annoyed every scientist in the world to the point that no one wants to read another word I've written, but this appears to be systematic.

I suspect that the grown-ups who have been doing the heavy lifting on this are at home where they have other responsibilities like children and methodically redoing their bathrooms with 1 cm square tiles (what I assume tenured PIs do in their spare time)

Editors -- the starter's are out -- time to go to your bench!

Seriously, there are people out there who may not be as buried in pressing responsibilities -- or haven't reviewed 10,000 papers in their lifetimes already. Proof? Someone set up a Google Document of people volunteering to review! (...and it's not just for postdocs...)

If you have ever wondered things like "hey -- why don't I ever get asked to review anything" because you'd love to. Now's your shot!  You can add yourself to the list here!