Friday, September 17, 2021

AlphaTIMS -- If you have a TIMSTOF you'll use this all the time!


Getting a good snapshot of this great tool was tough (and file name dedacted just in case it matters to my collaborator).

TIMSTOF files can be gigantic and digging through them for peptides that you know are there can take a really long time. Apparently some people at Max Plank noticed this fact and decided to take a swing at fixing that part with! 

Have you done enough reading for this lifetime? You can just download AlphaTIMS at this Github

Lots of folders? 

There is a "One Click GUI" and the software is pretty straight forward. You can filter your data by scan number, retention time, quadrupole settings, 1/k0 value, ion intensity, etc.,

What you'll find is that it's really easy and extremely fast to hunt down specific ions in your data. I'm still a little unclear the differences between the m/z index isolation and quad isolation functions. Wait. Nope. I just needed to type this (and maybe espresso to do espresso things) for it to click in my head. The quad filter is for the literal quadrupole. The m/z index is just my m/z view window. That's why the former doesn't do anything if I don't have fragment ions selected. 

You can also use this to kick out data as HDF, CSV or MGF. 

My understanding is that this is just the beginning of a whole bunch of new tools for this system that are in various levels of development, but I'm using this one constantly now and just looking forward to seeing what is next. 

Wednesday, September 15, 2021

SureQuant-IsoMHC - absolute quantification of antigens in patient samples!


MHC/HLA peptide studies for global proteomics generally start with "we cultured approximately this many cells.... generate enough peptide for a single LCMS run" 

This works for pure discovery and it still seems like we need to get the discovery aspect right, but for patients we need to crank up the sensitivity and here is a really impressive new way of doing that

SureQuant uses internal standard heavy labeled peptides to trigger focused targeted analysis. I haven't used it myself, but I also hadn't seen a reason that screamed to me that it was a necessary tool until now. High resolution absolute quan of HLA peptides....from a punch biopsy?!?!?  I strongly recommend checking this out. 

Tuesday, September 14, 2021

Comparative solutions for genotyping of human hair with LCMS!


Ruhroh, Reorge. Glendon Parker is still out there trying to rain on our free proteomic data parade in this new JPR paper

We've been doing some classical proteogenomics and getting the "publicly deposited" genomic data for it was a huge hassle. Justification forms and waiting months and we were under a really strict deadline for the project that was imposed by the people who had the genomics data. Longest part of the project? Waiting to get approval for the whole exome sequencing data.

You have to make sure that someone isn't going to use that 250 GB RNASeq data file to extract personally identifiable data from the patient for nefarious purposes. The 24 high pH offline fractionated normal and tumor data from that same patient? Pull that down as fast as your internet connection will allow. 

Could you identify that patient by the single amino acid variants you can find the proteomics data? 

....let's umm.....go with...wait! change the subject!  (Dr. Parker, that's enough from you and your group. I don't want to wait 2 months to download every .RAW file from every preprint. I'll forget to do it!) 

Look, we are going to have to tackle this at some point. Either the genomics people are being crazy paranoid about personal data, or we're being lackadaisical. 

I'm definitely being that (did I spell that right or is the spell check off?) last word, because I sat in on a webinar the other day and someone showed a slide that I recognized as a list of single amino acid variants that you can see in my personal plasma that are confirmed by my personal whole genome sequencing data.  I've got some plasma proteins that look downregulated vs pool in some analyses because of the variants. I use the slide to point out issues with extreme ratio quan in some LCMS tools and why we need to think about variants and I appear to have shared that deck a lot. 

In a world where the terms "pre-existing condition" and "life destroying medical bills" (still the #1 cause of bankruptcy in my country where people are dying at a really depressing rate because they are using expired or black market INSULIN because they can't afford the real stuff) maybe we wouldn't actually care about what of our personal data is out there in the world. 

But if we actually care, we might want to actually care about all of it. Not just the stuff that you need access to an HPC cluster to properly process. 

If you do want to identify someone by their single amino acid variants IN THEIR HAIR, this new JPR study will tell you which hardware solution to use for it and how to best set it up. 

Monday, September 13, 2021

Inactivating coronaviruses!

Remember that COVID thing in the spring of 2020 that was such a pain in the ass? Holy cow, how many of us needed THIS paper? 

Thankfully, it turns out that those things aren't all that hard to deactivate, but what a great reference to have in case a coronavirus becomes a problem again!

Saturday, September 11, 2021

Scale up (or go to lab less!) with 384 well plates on your LC!

You probably already knew this, but until I didn't until recently. Every EasyNLC that I've ever used has been set up with 6x8 autosampler vials by the previous owner. I guess I've never had a new one, which... come to think of it... might have something to do with why I've been so mean about them, they're pretty finicky about being abused.... oh well... well, if you didn't know you can set them up with plates!  

Even if you use long gradients I bet there have been times where you've made tough decisions with 6x8 about either going in over the holidays or skipping some things because 48 x 3 hours still isn't all that long. We've been using 96 well plates, but with 30 minute gradients I've made hard decisions more than once. 

ENTER THE 384 well plate! 

As always don't take my word for any of this, where did you get this information? The internet? 

This is what I'm using

384 well plates (there are less expensive options, but there is some weird virus thing still disrupting stuff, some other 384 well plates that I've ordered for sample prep have been backordered for 5 weeks)

Webseal tape

Your EasyNLC probably already has an option for 16x24 plates. Woo! You're done! 

(I'm joking! Stop! You'll break your needle! I was going to put this below the picture but had a fit of conscience.) 

My A1 position was around B2.4. I don't know if I could have ran it without calibrating first, but if I had, my needle would have pushed right into the space between B2 and B3. 

You'll need this manual if you have an EasyNLC1200 (WARNING. DIRECT PDF LINK.)

You'll also want a flashlight (or a fancy phone with a flashlight. I made my Siri British somehow and I have to say "Siri, turn on the torch". In the basement here it says "working on it" then "working on it" then "something went wrong" in a snarky Alex Kingston need a flashlight.

As shown in the top picture, you need to remove the steel plate on the left side of your LC. The 3 screws are snap screws. Turn them counter clockwise 90 degrees and they'll pop free and the plate will come off. 

Install your 384 well plate (without tape on it, unsealed!) and start the calibrate procedure (the one in the middle of the screen)

The calibration isn't a complex one like an OpenTrons you just need to position 2 points. The bottom of your A1 well and the bottom of the well at the complete opposite corner of the plate.

If you touch the A1 position on the screen the needle will go somewhere near there, now you need to manually locate the bottom of the A1 well. 

You can see the A1 well perfectly so that one is easy. Use the extremely European touch screen controls (needle in/out is not into your well, it's into your instrument. you'll see what I mean) to navigate your needle to above the A1 and down until it touches. 

Now -- Take a picture of your needle coordinates. My A1 was this, I think, but what I care about is the depth of the needle in A1 because you can't see into the far opposite well (I don't know what that one is called yet, I only filled my first plate to N6) 

NOW you can capture your coordinate! Hit the green well above the opposite A1 and repeat the process. Get the flashlight. If you have bifocals, get those too! 

Without having the picture of the depth of A1 this will take forever because the plate will move just a little and you'll freak out that you hit bottom and will be afraid to go further. I verified that the bottom was just about perfectly the same on both sides by raising the needle 0.1 arbitrary units and finding just a little wiggle room in the plate). 

Now, I'll probably keep using this tape that I linked above, there is a very satisfying "POP" sound when the needle goes through it, but it does leave a permanent hole in the top, so you probably can't inject more than once out of a well unless you've filled a blank. Anything you worry about evaporating will probably evaporate, other than that, I'm up to L and the QA sample looks legit. Everything is working.

Oh crap. Almost forgot. 

You'll need to adjust your plate in your instrument configuration so it knows you did this. On a Thermo instrument it's the little double gear thing probably (which is now an App in Windows 10. I hope it makes this 1990s program feel totally hip or something).

On the Brukers it is one of the circled things. I always try them both first. 

You'll need to just let the queue know that you've got a 16x24 installed! Now you're done! 

Friday, September 10, 2021

Carrier channel math on Orbitraps! Something proteomics people agree on!

There is so much important foundational work being done in single cell proteomics right now! I know several people who are very interested in starting to do this once "it's all done" and this is the stuff we need to get through. 

1) has some of the prettiest plots I've ever seen for proteomics data 
2) started in a completely different and really smart way to work out carrier channel ratio compression (using swapping TMT126 chanel for TMT0 to get some sense of ground truth.)

And what's really impressive is how consistent every study that has looked at using amplification via (SCoPE-MS or BOOST or whatever terminology this all lands on in the end) has been in the end. Now, this is the most conservative estimate to date, but everything has been pretty much in the same ballpark. There is a single order of magnitude between the lowest estimate and the highest, we're somewhere between 20 and 200).

Always worth considering here, and I apologize if I'm starting to sounds like a broken record (whatever that means) but every study so far has been on an Orbitrap, with only one group using the bigger D30 Orbitrap and everything else using the D20. Not to oversimplify the physics involved, but it's not just coincidental that the larger Orbitrap measurements have biased toward higher carrier. 

We've known all along that while the Orbitrap is one of  the single most awesome things that has ever been invented, the instrascan linear dynamic range is NOT why you buy one. You want big instrascan dynamic range that's linear? Get a QQQ. Everything else falls in the middle. 

Back to the paper: 

Another really good paper I'll be referencing all the time has done the carrier ratio suppression math and just to flag why I'm so impressed by them getting to the same conclusions: 

We're a 20+ year old field and not one single one of us can digest a sample the same way. Proof in point? 

Paper earlier this year? 

1:50 trypsin with overnight digestion at room temperature

10mM DTT for 30 min at 56C

55mM IAA for 45 min at RT

Some other changes: 

In Claudia's new paper we see something slightly different (surprise)

1:100 trypsin overnight at 37C 

50mM DTT for 30 min at 37C

40mM IAA at room temp for 30 min

Does this change anything? Probably not, but if 2 proteomics people from different labs land on the same conclusions about anything it's really impressive. 4 or 5 studies coming up with very similar conclusions? Maybe this current generation of proteomics trainees will fix the rest of it! (I'm super pumped they both used the same alkylating agent.) 

Thursday, September 9, 2021

Bispecific antibodies -- How to ruin a cancer cell's day with a neo-antigen!


I guess this isn't new news, but it's news to me! Hard copies of Science kind of arbitrarily end up on different coffee tables and I don't always get to flip through in order. This overview of the field and the promise it has is from March is a solid and approachable read

Importantly for me, it helped put this other paper I've been thinking about here and there into context and suddenly -- it all clicked. (Don't you love when those disparate neurons realize they're all related and it all falls into place?) 

You know how we're all called on from time to time to identify those awful MHC, HLA, endogenous peptide, surface neo-antigen things? If you have you've probably been at a point where you've struggled through it all and handed over a list to someone and:

A) You never hear from them again and you might wonder what they are going to do with it


B) (Way way worse) they ask you something like "okay, which one should I spend a lot of time, money  and resources validating?"

In this study you see behind the scenes and get a glimpse of both answers! 

This group prioritizes the neo-antigen targets through a process called MANA-MRM! 

Then they build a bi-specific antibody to the neo-antigens and totally f' up some cancer cells with it! 

As much fun as it is to bicker about trypsin concentrations and p-values, I will admit that I get a lot of inspiration from the couple papers a year that show LCMS can address biological or medical challenges. 😇

Wednesday, September 8, 2021

Isotopic Distribution Calibration!


Holy cow. I'm just going to leave this here until I have time to come back to this. If you're solely doing shotgun proteomics by LCMS this might not be the most interesting thing you've ever seen.

If you're from time to time digging all the way into your data to determine the difference between a PNGase cleaved glycopeptide or a citrullination and a C13 isotope or 4, or you've wondered things like:

 "why can't I use isotopic distributions at 700,000 resolution to determine if this is really pomegranate juice because of the different carbon fixation mechanisms used by different plants"  (hint, it still isn't pomegranate juice, global production is still only a small fraction of global consumption)

 or attempted to use isotope shifts to determine how old the material is in a drilled core geological sample is

or tried to distinguish between natural and synthetic compounds 

You've probably been extremely annoyed about the inability to calibrate your isotopic distribution and maybe thought of crazy ways to do it yourself and then given up. 

If any of this applies to you: 

Tuesday, September 7, 2021

NeoVanquish -- Dionex finally released a new NanoLC!


When Dionex acquired Thermo Fisher Electron almost 10 years ago they needed to spend a lot of time celebrating. I understand it. If you were the 8th best company in a field of 6 manufacturers and you somehow acquired a Fortune 500 company that had a year over year somehow sustainable growth of over 20% due to a strangehold on an entire emerging industry (proteomics?), you should probably take a bottle of tequila into your office and celebrate in private until someone realizes what just happened makes zero sense at all. 

If no one does figure it out and 8 years later you find yourself still in ownership of said company (that... longer has such a strangehold on the industry...but I'm sure that's completely unrelated...), maybe you should use all those resources and roll out your first new product in a decade! 

And here we have it! A blue and black NanoLC with a touchscreen! 

Honestly, the specs look pretty impressive. They might even be better than this NanoAcquity we found in a closet that I have to run on a laptop with Windows XP for some reason.

Monday, September 6, 2021

Working in Tandem -- The first annual symposium dedicated solely to multiplexing proteomics!


I'd promised to get the word out on this, but then I was being totally awesome and then had to get surgery for being too old to be pull off being awesome and I've mostly been doing my cantankerous old mass spec wizard act, albeit with more over the top swearing than average for my peers.

Now that I'm mostly back in action -- check out the line up for this killer TMT symposium

It's next week (!?!?) and the speaker lineup is seriously stacked!