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....


.....to 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 way...so...I 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

or 

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... hmm...strangely....no 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!  

Tuesday, August 31, 2021

Proteomics goes after the muscle cell secretome!

 


The introduction of this great new study has all sorts of cool biology in it in terms of how skeletal muscle cells and exercise end up secreting (myokines?) that lead to all the positive things our physicians tell us they do. 


I'm most impressed with how clean and smart the experimental design is to help add new information to how a central tenant of middle school health classes actually works. Cells grown in culture are stimulated with different things and the secretome is sampled from the cultures. An HF-X is used for the LCMS and MaxQuant for the data processing. The logic of the study is really clear and well executed, and the figures clearly drive home each point the authors make. 

Friday, August 27, 2021

PepSep -- the best chromatography doesn't have to be the most expensive?

 



I held off on putting up a post about PepSep until I knew I was personally stocked up on columns. 

No disclaimers here -- PepSep doesn't give me anything for free, or even a discount over what is on their website. I've never met the mysterious person behind the best columns I've ever used. 

If you use an EvoSep or have a Bruker, I'm going to guess that you have seen columns that look exactly like these, because I strongly suspect this is the supplier for both. 

Even better than buying from the source? How could that be? Well, what if you could use a column configurator to assemble your dream column from a very straight-forward list of options? Insanity? 



What if you want 3 25cm columns with a 50um internal ID because you are slooooow flooooow weirdooooo and even want smaller bead sides and you want them to plug up directly to your instrument? 

You can do this here

3 columns for only 1475 squiggly things? Try beating that! 


Tuesday, August 24, 2021

Too hot to handle --new antibiotic peptides...from ghost peppers?!?

 


I haven't paid much attention to antibiotic developments recently but when I was in grad school (in a microbiology department) the fact the world was just about out of antibiotics way back then was a pretty big deal. 

Want to go hunting for some new antibiotic peptides in material you wouldn't necessarily suspect would have them? You can't go wrong starting with what is detailed in this great new study!


Ghost peppers have antibiotic properties? I'm sure you're familiar, but this is the crazy hybrid pepper that typically checks in at over 1e6 scoville units. A typical jalapeno is 2,000 to 5,000 depending on how badly you mistreat the plant and different habanero varieties are in the 1e5s.

Before you get too excited, I started this post with the chart above which demonstrated the antibiotic properties each HPLC fraction had against E.coli because I don't think that Taco Bell uses the late eluting fractions. This study shouldn't be used as justification to start another petition. 225 supporters? I'm sure this one impressed a giant global corporation. 


Jokes aside, this is a really well executed study on how to hunt down antibiotic compounds from an extremly complex and largely uncharacterized starting material. This team worked out inhibitory curves against at least 5 different bacteria, both gram positive and negative. And progressively worked their way to enriching and characterizing a large endogenous peptide responsible for this activity. The LCMS work was performed with a SCIEX 5600 and Q Exactive HF-X and the files are up on ProteomeXchange via PXD024605 and -- this is something super cool I wouldn't have thought of at all -- the plant that produced this pepper has also been deposited at a greenhouse at UNC! What a great idea for both reproducibility and in case this specific plant was a one-off, I guess? The Ghost pepper is a recently generated hybrid plant product and plant genomics can be super whacky. 

This blog doesn't have a lot of rules, but one is and will always be: if Randy Savage ever said the title of your paper in an interview it has to be linked here. 

OH YEAH. 

Monday, August 23, 2021

Revisiting the ion trap!


Hopefully you didn't catch my 15 min speed rambling at SCP2021, but one of the things that I tried to emphasize was the value of when an ion actually makes physical contact with the detector of a mass analyzer. 

It happens in most hardware, including the long forgotten and often maligned ion trap. 

Is it time to revisit this relic of ancient history? There might be an argument here


This ion trap appears to fair quite well in these comparisons even considering where it is placed within the instrument that they are using. Not that the architecture of a hybrid instrument has anything to do with anything when they accomplished 100% ion transfer efficiency a while back. 



Sunday, August 22, 2021

Ice-R lecture is up and the paper is out!


I'm still excited by the potential of IceR even though I posted the preprint pretty recently. You can find it here. A slightly modified version of the study is now available here.

For a walkthrough of what makes this study so very cool, you should be able to click on the video above, or watch the video at the singlecell.net YouTube channel here

The outside bioinformatics people are starting to drop in to see what is happening in proteomics.  I swear, 1/3 of the new questions this year on r/bioinformatics have been about proteomics. I can't overstate how noisy (and HUGE) the data that they've been working with is compared to the stuff we generate. I think we're going to see transformative developments on the informatics side very very soon. 

The flipside is that our bad habits are going to be way out in the open. I've witnessed some shock and horror about our seemingly random use, as a field, of two different alkylating reagents that differ in mass by a just about the difference between a carbon-12 and carbon-13 and the common lack of metadata that will tell someone who pulled down 2,000 LCMS files which file used which.