Thursday, November 30, 2023

An AI helped make this month's JPR cover!


Another bad week for the blog, but I've been under multiple deadlines and I'm sort of tired of writing. But here we are at 3am typing in the orange box instead of working on things that are due in like 6 hours. 

This probably isn't the first JPR cover generated with assistance from an AI? I don't know, but this one used help from the DreamStudio AI. 1,000 iterations cost $10 and this one didn't even use 100 of those. will generate some whacko nonsense if you don't use the right terms. And if you give it text it does not know what to do. Is that kanji? Or is it just random lines? Have it generate pictures, fine tune them and then add everything else. Is that yogi the bear doing yoga? That's funny, but not at all what I was trying to generate. 

One of my absolute favorite parts of this study is this candid statement in all capital letters. Why does a static gun need to be shaped in any way like an actual gun? As a lot of people know, I was very physically close to one of the most horrific mass shootings in US history.  It didn't do great things for my brain - which, to be fair, was sort of mediocre to begin with. Imma patent a static gun that looks like a pencil or one of those penguin like animals they have in Maine or something soon, cause working at 3am in a quiet inner city building holding something that looks like a f'ing gun deserves notice as a stupid thing. 

Oh yeah, this is the paper! Yet another single author paper? Weird. Okay, so there was a student who was supposed to do this study as their final 1-1.5 years for their PhD, but then that student just never came back to lab. So I had spent all this money on cell lines and reagents and hundreds of hours on training and planning a study I was emotionally invested in and I was a little annoyed about the situation. Instead of being all angry or sad or regretful for hours of my life when I don't have an infinite number of hours left, I cut some experiments from the study and knocked out the majority of the planned work over the Xmas holiday break by myself. 

Honestly, the coolest part of the study is the global metabolomics. Also - this might be the first published paper to use the SimpliFi Cloud based multi-omics data interpretation system? I've been a beta tester of this ProtiFi project since the beginning and it's super cool for sharing processed and interpreted data. For some reason I don't remember, I broke this study into separate SimpliFi projects. Here is one of them.  I hope we'll have something to directly import global metabolomics data from Compound Discoverer into SimpliFi. About got that node configured, it's just an abacus issue. 

We just uploaded some new data to MASSIVE that is way better than this, btw. The people doing the prep now are better at sample prep than I am. 

Monday, November 27, 2023

Single cell proteomics and single cell mass spectrometry meetings 2024!

In 2023 I was lucky enough to get invited to show off new data at every single cell proteomics and single cell mass spectrometry meeting of the whole year! There were a bunch...

My 2024 is up in the air and I can't really schedule anything after May, so I'll probably watch from afar this year, with a healthy dose of FOMO. 

As requested by a world famous single cell proteomics researcher in Canada, here is the breakdown of 2024 conferences. 

The first one of the year is clearly on this weird Spanish island that I definitely can't find on a map! 
JnM January 7-12, 2024

Placeholder here for iSCMS, which is in Europe somewhere! When I get a date, I'll add it. 

THE OG SCP conference is a week or so earlier this year. The Slavov group likes to have this before or after ASMS so people coming into the US can drop by this phenomenal little meeting on their way too or from the crazy big mass spec thing (which is at DisneyLand/world? or something this year)! 

 ESCP 2024 August 27th-August 28th 2024!  VIENNA! 

Sunday, November 26, 2023

YODEC - A strategy for biasing what you amplify in carrier channel experiments!


If you're using a carrier or boost channel ala SCoPE-MS, IOregano, or whatever you call it, you generally start with this beautiful pool of all your conditions for the carrier channel.

When using that approach you're basically only BOOSTing the most abundant peptides from your pool. And...maybe those aren't the peptides you're actually the most interested in! 

What if your carrier was depleted of some of the proteins you aren't interested in?  Could you see more of the proteins you do care about? 

This group reports that frog egg yolk (which...if that doesn't put a gross image in your head, you're a weirdo...) makes up 90% of their peptide signal and they've already analyzed every gross egg yolk protein and would like to see something else. 

I'm not clear on how they depleted the yolk proteins, but there is something called a "yolk depletion buffer" with 3 references that I'm too lazy to read (if I haven't whined about this on every post this week, RSV sucks, avoid it if you can). But their coverage goes through the roof on these FACs sorted frog cells. 15-fold more peptides than running it without? 

This lab has a bunch of nice instruments, and in this specific case they used an Orbitrap Fusion II using an MS3 based quan. The fact they used 15,000 resolution for the MS3 threw me for a loop, but in the supplemental (excel sheet) you'll find they used unit resolution TMT tags. Side note, some of the nicest plots are in the supplemental PDF because they didn't have to condense some really nice data visualizations to fit into PDF panels. Worth taking a look at for sure. Even if frog biology isn't your thing, this study certainly suggests that this method would have other uses! Who couldn't find an immediate use for ACDC-FAC(ACtin Depleted Carrier For Any Cell)? I could think of 12 right now.  

Anyway, solid study all around and I didn't know there were "Editor Choice" stickers for JPR papers and this one has one and deserves whatever that means! 

Saturday, November 25, 2023

Ready to SWIM? Sliding windows allow TIMS at ultra-narrow (GC, even!) peak widths!


In 5 years we'll stop getting new TIMS methods and will settle on the 11 or so that the majority of us will use. Of course, the inventors of their own will stick to them resolutely until their retirement, but about 25% of mass spectrometrists will have the acronyms straight.

Ready for the next one? SWIM time! 

One of the things that is super cool here is that they linked a Bruker (??) GC to a TIMSTOF Pro II by an APCI source, because. apparently that is something you can do....

So....I can't use TIMS when I'm using MALDI? But you can run TIMS in ESI, APCI, and DESI? Weird. But one of the big hangups for GC in high res is that high res is often too slow or too insensitive. So here you go, trapped ion mobility fixes both. My last GC single quad was $44k and a Pro 2 is a bit more than that.

Super cool study, though! 

Friday, November 24, 2023

Deep quantitative proteomics of human brain cells in culture dosed with CBD or THC! for something completely different and suprising! (honestly thought I'd posted this a couple weeks ago). 

Immortalized human neural lines doesed with mock controls or THC or CBD. These are the isomers in cannabis that there have been so many "miraculous medical discoveries" about in the last few years that it's pretty easy to assume they don't actually do anything at all. Though...I guess there is pretty good evidence that THC does at least one thing, so maybe my skepticism is just running amuck. Amock? Ummuck? Didn't this thing have a spelling and grammar check? Am I just rampantly leaving words out of paper titles? 

The quan is super solid, as you'd expect and 24 offline fractions aided by real time search MS3 means the coverage is superb. And - clear alterations in proteomic response and -- they're different between the two unmuck your first thoughts here. In at least one cell line in a petri dish you can change the proteins expressed by dosing with these compounds!

Thursday, November 23, 2023

Huge congrats to this year's US HUPO Association Award Winners!


Man, for years I kept forgetting that US HUPO was a thing. All the way up to not even knowing about the meeting when it was in DC and I was just working like 8 miles away. I only went to the one in Charleston because I was afraid that my friend tricked them into having the meeting there and no one would attend. Now I'm occasionally involved in US HUPO things and I'm on the mailing list and in Chicago I found out they give out awards to people - and they gave all 4 to people that I honestly think are good people doing important things. Who weird is that? 

Big congrats to all 4 winners! my interpretation of the way the world works is that we are not justifiedt to have all these people on the podcast and to send the bill for editing to US HUPO! 

Wednesday, November 22, 2023

A practical beginner's guide to bottom up proteomics - and a better mechanism for teaching -omics?


Okay, Martin's on this 2 times, but I always try to cut the entire author's list and this one took 3 pages of screenshots.

How do you get new trainees passionate about proteomics up to speed? Send them your 15 favorite academic papers? Give them a great textbook that was old when it was published? 

Are living documents like this what we should be doing for the next generation of scientists instead?

Honestly, this sort of thing seems to work out really well for the other -omics. I like the marked up copy of Hadley's book in my office, but it sure is faster to find things on his old webpage! 

I didn't even know you could build something like this through Github and - I do have to say - it might be intimidating for someone who is really new to this stuff, but it is a nice step in the right direction. 

Tuesday, November 21, 2023

Instrumentation at the leading edge of proteomics (end of 2023!)

Do you need a comprehensive breakdown of how proteomics technology has evolved to - right now? Do you also need to see the upper limits of what a citation manager can do? Do I ever have the preprint for you! 

750 references? I forget. Something absurd like that. I might be getting it confused with a text book that is in the queue for a post. 

Monday, November 20, 2023

TIMSControl 5.0 fixes many of my favorite things to complain about on TIMSTOFs!


WHAT??? Okay, so I'm starting to run out of things to complain about. I'll find something. Don't you worry. But this has driven me crazy for 3 years. While you're acquiring data on a TIMSTOF you can't edit your method. Honestly, for most things, just run the default method. But if you're trying to do something stupid, like - I dunno - simultaneously do proteomics and metabolomics - and you have to constantly screw with a lot of settings you have to make 10 or 20 instrument methods - then run your samples and evaluate them after. It's tough to realize you did something dumb in the middle of a queue and then fix it. 


You can also toss out your profile spectra and just collect your line spectra! That should cut some file sizes down (but - if you like profile spectra that might be bad for you later? Meh. All our hard drives our bright red and MASSIVE keeps telling me I'm 600GB over the limit. Smaller data sizes sound okay to me....

Flag when it's time to calibrate? 

For those of you who are in clean facilities with modern functional HVAC, this will be useful to you. On Monday my lab hit 94F (34.0C wasn't the highest this QE reported as "ambient") and due to all the dust, we have to change our captivespray filters every week or two, so reminders won't really be helpful for us. We basically calibrate every day or two depending on the environmental conditions. 

OH CRAP - IN BATCH CALIBRATION??? We'll use the fuck out of this! 

There was extra features I don't know about, but from a usability standpoint this is huge for us. 

Sunday, November 19, 2023

The social and structural yeast protein interactome!


Well...this wasn't what I was expecting to see today! 

4,159 GFP tagged proteins (all of the yeast ones?)

Yeast grown and lysed (and digested?) in 96 well plates.

22 minute EvoSep runs (EvoSep 60, rapidly becoming my favorite LC method). 

Beautiful website!

Website may have a glitch behind the scenes? I can't look at all of the proteins. The pull-down looks like it might have a maximum number of entries and it stops part way through. Mostly putting this comment here for the authors to maybe find.

I think we'll start to see an avalanche soon of studies where people think things like - I only need 0.1% of the material for this experiment that I used to. And...I can see all the proteins in this organism in like 10 minutes. Let's do something crazy huge! 

Friday, November 17, 2023

ProteomeXchange got a glow up and provides deep insight into proteomics today!


I went to look and see if I remembered to make some things public and - BOOM - way nicer ProteomeXchange front page with all sorts of really cool insight into the state of proteomics today!

Just zooming in - check this out! 

The majority of publicly available data is human, by quite a margin! Honestly, I figured mouse would be a lot closer. And I'm sad to see my old nemesis scumbag arabidopsis is number 5. 14 years out I've almost recovered from Arabidopsis saturation from going to a land grant agriculture university. 

It's novemeber of 2023 and we've got to get some submissions up! 2022 was a bigger year for proteomics? Some of y'all are running 3 minute gradients!

Zooming in again! The great Q Exactive is - hands down - it isn't even remotely close - the instrument the vast majority of publicly available studies have been ran on. 

And - get this - some of those other blocks are where someone actually put in the model of Q Exactive. The HF is second place! with 3,500.  The QE Plus has over 1,000 studies and the newest/last Q Exactive, the HF-X has almost 800. There are a bunch of those out there churning through samples - so I expect that number to climb for the next 5 years or so. 

The Exploris just misses the top 10 as 11th and the TIMSTOF Pro checks in at 13th. TripleTOF is the only not Thermo vendor in the top10 at 1500 studies.

The TIMSTOFs are definitely diluted a little because they drop a new model every 6 months to 1 year. For example, none of my group's work is counted in the TIMSTOF Pro numbers because we're TIMSTOF Flex and SCP. 

Super cool stuff! How often can you get this sort of insight into an entire field in just a glance. Bravo to the consortium for this smart new interface.

Thursday, November 16, 2023

Data out of the Apollo cancer moonshot -- Multiomic analysis of ovarian cancer!


Whoa. Okay, sometimes it comes up - "hey, have you seen anything out of the Apollo/Cancer moonshot thing yet?" We keep hearing they're doing HUGE multi-level projects and you'd assume those take a really long time. Turns out, I just don't keep up with Nature Genetics, because this huge, beautiful ultra-impressive study has been out for a while.

Genetics? "You mean that stuff we make fun of?" Yeah....but if you've got a cancer that tends to end up with nonfunctional mechanisms like double stranded DNA break repair because homogenous end joining just doesn't work anymore, you need to investigate this with genetics technologies. When DNA breaks checkpoints should activate to stop the cell from dividing. right? Then depending on how the DNA is broken you'll either activate HR (homogenous repair) or NHEJ (non-h end joining repair) and probably other processes that we didn't know about when I did my first postdoc on DNA repair proteins. 

This study goes in depth into end-stage ovarian cancer from a multi-organ/multi-omics approach from autopsy samples. It is as comprehensive as it is depressing. My dog, there is so much heterogeneity in how these patients died.... looking through it, you feel like true personalized medicine is the only answer to keep someone alive once cancer gets to a certain point and the level of the study depth that we'd need to have for each individual is at a level that only Elmo Musk and Jerf Bozos would be able to afford it. 

But the technology is here, and if you make a monumental effort you can use the technology to get the complementary insights to pull it all together. Seriously awesome and beautiful study even if it suggests how far we have to go. 

Wednesday, November 15, 2023

Proteomics Journal Page Charges - the 2024 list!


I've got a minor crisis submitting to ACS journals since they're over my credit card limit ($4k, or $3,750 with our institutional discount - I can't charge over $3499.99). This is the same for JASMS, btw.

I can afford the page fees - for real - but since it has to go to PO - they'll issue those funds when they...feel like it..... And if I have the audacity to ask when that is, they'll push it to the bottom of the pile. My OA fees from a paper in JPR accepted in January were paid in August.  Update: Because I'm citing papers stuck in OA release purgatory I was able to get my OA fees through purchasing thanks to my front office going above and beyond in pushing these through in just 6 weeks (thanks Debbie!!)! So I don't think I'm currently in collections with ACS/Rights Link until we get the proofs out for the next 2 papers...


Are there places I can submit my data that fall under my credit card limit? (I am required by my funders to comply with open access mandates).

What about those nitpicky nerds at Molecular and Cellular Proteomics? $2,800!! (after the 2024 increase). Hmmm....okay....maybe I should stop calling them nerds all the time.... Now...the reason no one ever cites MCP papers is that THERE IS NO "CITE THIS PAPER" BUTTON ON THE FRONT PAGE OF THEIR ARTICLES, so you have to manually type all the info into your citation manager? I honestly don't know how other people do it. Like every normal person I just don't cite MCP papers. I ain't got a week to find out how to cite it. There is always some review somewhere that I can cite instead, probably at a journal where it is actually easy to automatically add that paper to my citation manager. 

Wiley Proteomics is free to publish, but $4430 for open access fees. Ouch... I always forget that one is a thing. Not a winner. 

Elsevier has a proteomics journal...? Of course they do! It's called Journal of Proteomics. $3450. a pinch, I guess.... they probably require that they encrypt a folder on your hard drive if you do submit to them, but...meh....

Clinical proteomics appears to still be in business...? Yup, still there! And OA is $2490

Proteomes has always been the most affordable option, once you figure out how many Swiss Francs are in $1USD, but - like everything else in the world, it's creeping up as well. After conversion today $2032, but that's before institutional discounts, which are typically considerable. They also don't check them well. Since I still have my grad school email and they get a bigger discount, I just pay with credit card under that email address. That's a joke. I wouldn't do something like that to just save like 300 Swiss Francs, whatever that turns out to be in real money. 

Worth considering, Scientific Reports likes proteomics data and it's $2490. According to them, it is the most cited journal in the world, so it sounds like a good deal even if the impact factor isn't a positive number. 

I read a lot of preprints and assume most people do, but a lot of people don't count them toward your productivity. I can't even type them into the reports that people examine at my work to see whether I still have a job after May, so we've worked out this equation at home where we assume each 1 published paper ~ 5 preprints. 

Tuesday, November 14, 2023

VISUALIZE DIA-NN results in Skyline! 2023 MVP level tutorial by Brett Phinney!


What is THE biggest problem with DIA-NN? By far? My vote would be that all I get is this sheet and I have to believe Neurotic Network things that it is real. I can't check my peak quality or my fragments because all I have is a bunch of text files.

What if you erase that limitation by using another 100% free -to - you incredibly well supported, validated, respected and ultra powerful software - like Skyline???

Dr. Brett Phinney, Director of the world renowned UC Davis proteomics facility to the rescue. Check this ridiculously great video out here!

On that topic, all of the videos from the UC Davis proteomics course are also online. You can find these here.  

Monday, November 13, 2023

What does an LCMS proteomic run cost? Breakdown for my lab.


This weekend on BlueSky we spent a lot of time talking about what proteomics costs today per sample and I thought I'd do a run down of what it costs our lab to run a sample. 

Further clarifications for the most popular post in a few months (eek)

This should be called. "What is the bare minimum expense for a PI with 20 years of LCMS experience to complete a single shot LCMS shotgun proteomics experiment at a depth of approximately 5,500-7,000 proteins measured per cell." We are not a service center or core. We are not regenerating our costs in any way. This is the minimum cost for us to do this aspect of our research. 

EDITS AT BOTTOM! While I was driving in through the Thunderdome today and should have been paying attention to one of the most dangerous stretches of road on the entire east coast, some critical dislaimers came to mind - then when I saw the popularity of this post I felt like these were important enough to hold up my breakfast. 

Our biggest costs, by far, are our instruments. 

The TIMSTOF Flex we purchased in late 2020 (when I arrived at Hopkins) was a ridiculously good COVID panic deal from the vendor. No joke, I think it was $500k less than what I was originally quoted. 

This year's lease payment on that instrument is $188,000 or something. I might be rounding it one way or the other but that makes me think it is about $500/day for it to be here.

Mike MacCoss estimated that his people's salaries are a lot more than his instrument leases. I suspect people are a lot happier to go to work at University of Washington because I can take my salary and two other people's in my lab and those don't add up to our instrument lease. It's close-ish and below I'll use it in the math to assume the same thing.  

I should know for certain, but I think because of the laser on the Flex our annual service is higher than something like a TIMSTOF Pro. I believe it was $60k. I probably won't be able to afford a service plan in 2024 and the 2023 one has been a poor investment because literally nothing has broken on it the entire year. My laser went over the "maximum number of pulses" limit in 2021 and it still passes QC. 

Since we're going to cross 8,000 LCMS runs (and, obviously a LOT of MALDI runs) any day it is pretty clear we do just about everything with this Flex so I'll just focus on it for now. 

Then software:

SpectroNaut increases in price this year, but last year my key was $7,000 academic

Proteome Discoverer has some really weird pricing structure stuff now that I honestly don't understand, but it was $2,500/year to keep the Protein Center nodes active. This year we had to purchase xxx number of hours of Jackalope even though we can't use it so I'll just round that to $3,000 so I can have software at $10k for the year. 

We prepare every sample with S-Traps. I prefer 96 well plates for prep and the last batch I ordered that ran out to about $3 per S-Trap. 

I just discovered some students in the lab are still using the complete S-Trap kits that come with reagents A/B/C/D and those come to around $20/sample. I generally keep those around to give to collaborators with cool samples who have never prepped a sample before so I need to do something about that. $20 each adds up to a lot. The spin columns are sort of in the middle. Maybe $8-$12 depending on how many you order? I might round it below

We have a paper coming soon where we investigate different trypsins. Honestly, it was a lot cooler on the poster Ahmed had at US HUPO. We found it less interesting as we moved into organs and plasma so it kind of went on a back burner. We estimated our typical trypsin usage to be about $7/sample

We use the EvoSep for every big project. Honestly, I'm getting closer to a point where I don't know why we use anything else. If I've ever said anything remotely negative about the EvoSep, ignore it. We had some early issues with ours that were largely environmental. As one core director I respect a lot asked recently "Are your instruments outside?" Not quite. When it rains in our lab it might not be raining outside, but the temperature inside and outside are typically about the same. It's a tough place to be an instrument. 

EvoTips run us about $2. If we use IonOpticks columns they last about 2-4 weeks. If we use PepSep columns and we never take the column off at the ZDV union (they universally fail at the "NanoViper" junction) one of those can last months. The chromatography isn't nearly as good. However, if we do need to switch those, we can take one off and put it back on about 3 times before it fails. Tough to really estimate, but I'd put the Flex column consumption at about 1 column/month. So $800 for a PepSep or $1200 for an IonOpticks. Let's go with $1k/month. 

Quick math then! 

$500/day for instrument

$500/day for PI, 1 lab specialist, 1 graduate student

Service + software @ $70k/year + nanoLC columns at $12k = $82k lumped = $225/day.

For most things we're using EvoSep 30SPD which is 44 minutes LC gradient. We have a big study coming where we did 128 organs with EvoSep30 and 60SPD and the protein numbers are very close, but the number of missing values with 60SPD was a lot higher. With that many biological replicates, it's sort of a how much do you care at double throughput, but let's keep it at 30SPD.


$60 for EvoTips ($2/sample)

$300 for S-Traps (or $100 if we use 96-well plates, let's assume this even though it is actually higher for me right now) so $3 or so

$210 for Trypsin (or $7/sample)

And if we take the $1225 for lease,service,software columns and salaries for 3 of us that adds $1225/30 ~ $40/sample + $2 EvoSep + $3 S-Trap + $7 trypsin = 

$52/proteomic sample cost to me....

sort of assuming 24-7 round the clock nonstop operation with no QCs and no downtime. We're productive and ambitious, but that is absolute fiction. 

8,000 LCMS injections on the Flex in the approximately 1,000 days we've owned it says we're doing maybe 8 runs/day on average? (Minus MALDI time -- those imaging runs are typically like 1 day per slice, I don't want to do that math) 

At 8 samples/day we're $153/sample but the % MALDI vs LCMS is tough to break down. I'd like to think we're closer to the $52/sample. 

Or about $1,500/day when we're running full speed. 

What's missing? Biognosys QC standards (iRT), K562 from Promega. The big data processing PC that we bought when I got here for $12k? That is full and sorta slow by today's standards and could use a replacement in the near future. My monthly payment to IT to protect everything. The air filters for the captivespray source (clean labs go through them a few times a year, we have to change them every 2 weeks or so). Calibration mix. Adds up, but it is honestly sorta trivial compared to the other things. 

Obviously this gets a lot better when the Flex is paid off next year, but that's where we are. So if someone offers us $2/sample on a grant to do a project.....I might not want to be on that project....

(Not my example - someone else was offered $2/sample....not joking...)

New disclaimers! 

Okay, so - please keep in mind that the National Institute on Aging and National Instutes of Health Center for Genetic Medicine pay for me and the people in my lab. One of the things they also pay is for about 64% of every dollar to go to lab indirects. The samples that we're running are for these projects. Some other funds trickle in other places and for a pending collaboration with a company, a little over 76% of every dollar will go to overhead/indirects...honestly I've tried to talk them into working with someone else, but they like me. That's a big deal because the day these overheads stop coming into my university, I'm unemployed. So is my team. 

As such, we're doing research here on what we found interesting and important enough to write some grants on and we're hugely enormously forever grateful that someone said - WHOA, you're right, that is important, buy some stuff and hire some people to do it. (Thank you thank you thank you, you know who you are and you're on every slide deck from the lab). 

As such, even if we were running 24/7 (which we aren't, but we try really really hard to use every minute) any project that isn't - you know - actively keeping me and these cool young people here employed - is more than detracting from these amazing gifts from the government and corporate collaborators provide so we can work on stuff we hope will make the world a better place.

When I say a minimum of whatever above - that is for me to do work on these projects that I'm genuinely privileged to get to work on. If you offer me $52 to run a sample for you that I don't think is interesting, I'll show you that our wet lab has 3 separate doors (we're in a corner, so there are plenty of ways to leave!)  On a good day I'll provide you some guidance on good cores. The one 3 floors up from me is awesome. They sure as hell won't do anything for you for $52 because they have to make up all their costs and - I hope - they pay their people a lot better than I'm allowed to pay mine. 

That's a rant, maybe. But if you're reading this and thinking you'll leverage this to get some cheap proteomics samples ran because you know what one lab's rough math is for what it costs them to run stuff on their own instruments, you aren't very good at reading.

Someone at Harvard (hi Amanda!) brought up the training aspects. Oh yeah! Okay, so I've been running instruments for 20 years. And we still pay for instrument and software training and apps support. Vendors might argue I beg some freebies, but we've definitely cut POs for training.  And -- I had a student rotate through the lab a couple years ago who killed 3 nanoLC columns on her first batch of samples and her rotation resulted in one figure that might make a paper when I write it mid-2024. Not calling him/her out, but training can be expensive. Another example - we had the TIMSTOF Flex from just before Xmas of 2020 and the first data to make a publication didn't come off the instrument until March. It takes a lot of time to learn how to use one of these things. The big NIA project I mentioned that pays for most of this has over 1,000 separate LCMS runs associated with it. We probably will only upload to MASSIVE around 400 because we just got way way better at diaPASEF in the last year and nothing new comes from the old samples, just making us look like we didn't know what we were doing (mostly me).

Since this was a lot of fun, maybe I'll do the metabolomics one later. I don't know that math. I do know we spent like $6k? $8k in pure standards? It was a lot, but I'll look it up. 

Sunday, November 12, 2023

Incorporation of a virtual proteomics experiment into undergraduate curriculum!


I LOVE this idea and great recent paper! 

Now, if you've been to some vendor sponsored workshops recently you've probably heard things like "with our complete solution you don't need experience to generate great proteomics data, just give us >$1e7 and find someone who can read and you'll be rich". 

I'm not sure how much I can say about this, but recently a vendor said something like that to some people who were stupid enough to believe them -- they failed (surprise!) and they sued the vendor for >$1e8't work out for the vendor....

What this paper suggests is that maybe - just maybe - we can start inserting proteomics experiments - and associated understanding into undergraduate courses! Proteomics is increasingly ubiquitious. Informed people are going to make better judgements, and come up with better experiments and this amazing little study suggests we can start making big strides right now! Then you could sort of be the compromise between - "don't worry, our instruments literally run themselves lawsuits" and the whole thing happening right now where there are lots of proteomics jobs and there aren't anywhere near enough people to fill them, so they're getting filled by people who think "proteomics" is that ELISA or Western Blot they ran in their third year of grad school. Maybe if we had people graduating with some basic skills in the area both crises might just fade out!  

So this paper is 100.0% recommended! 

Saturday, November 11, 2023

EuBiC Winter School registration is closing soon! Come to Winterberg!


I have a bunch of conferences on my career bucket list and it's time to check off EuBiC Winter School! Early bird registration is closing really soon and then your stuck with late bird registration.

Check out the cool site they have with feedback from 2022 Winter School! 

I tried and failed to get to Winter School 2019 - some evil people allowed our government to shut down so long we didn't have air traffic controllers and I literally couldn't get off the runway. 

Can't wait. Can't wait. Can't wait.

OH YEAH one thing that isn't on the program yet is that Ben Neely and I have a workshop on how to distort social media to the benefit of science. As part of the workshop we'll walk you through how to make a science podcast in the hopes that you'll make a better science podcast that we'll listen to so we can stop making our mediocre one. 

ON THAT NOTE - If you missed the US HUPO Proteomics Quan Battle Royale. We ripped the audio from it to make a new podcast. You can listen to that where you get podcasts. 

I just pulled this up -- the bar is very low for podcasts in Australia! You could come to our workshop and take this over 3 days later. 

Friday, November 10, 2023

Circadium protein atlas (in mice) across 8 organs!


Circadium rhythm (is that spelled right? I bet it isn't. ryhthm? meh...) has been heavily investigated by transcript abundance, but we all know that measuring transcript abundance is 

This group knocked out two of the main circadian yrhtymhy proteins and then actually measured the things that people care about --> over 11,000 proteins! 

They made an easy resource that is almost as hard for a dysgraphic guy to spell at rhthym. You can find it here:

I have to admit I don't understand what the numbers mean under every protein that I've looked up in it, but I assume it'll make sense to people who know about circadium thrymrhths. 

The quan was done by isobaric tagging and a crapload of fractionation with LCMS on an Orbitrap HF or HF-X system. Curiously, the HF doesn't appear to have gotten the "TMT mode" upgrade to allow 45,000-ish resolution, but the HF-X did. So part of the files were analyzed at 60,000 resolution MS/MS while part were done at 45,000. If the authors see this, I was pretty sure that was a free upgrade? In my hands the HF and HF-X pretty much perform the same unless you are going to low loads. I think that at the amount on column here if the resolutions were matched the data would be about identical between the two, particularly when using a 100ms ion injection time. 

I wonder if they traded in the HF part way through the study and got an HF-X?  I need another coffee. I don't remember what I was even typing about. Oh, and they used a 0.7 Th isolation window in both experiments. 

Data processing was done with Proteome Discoverer and I think they really should have provided a citation for it, like this one. 😇 

Holiday peptides?

Sleepily just walking by these boxes on the counter has caused my brain to glitch every single time. 

Thursday, November 9, 2023

Great. Sulfo-Tyrosines are important? Here's how you detect them!

 I think the picture above encapsulates the problem pretty well, right? There is a 0.01something difference in mass between a phospho- and sulfo-tyrosine.

Better to just ignore that either of those are things and move on, right? Unfortunately, a this new paper points out, the sulfome is important in viral infections and even knocking out sulfo-transferases in mice makes them skinny and have poor vision....

...and we don't have any idea what the real breadth of the sulfome is. How many proteins get "sulfomed" (sulfo-ded?).

Until now! 

There is a lot in this paper. But they basically work out a modified IMAC that can preferentially enrich peptides with sulfated tyrosine residues using acetic acid to charge them and titanium or zirconium IMAC enrichment (go acetic acid!) 

Also - even ignoring the sulfonation stuff - how cool is it to be able to enrich modified tyrosine peptides without one of those awful anti-phosphoY antibodies. I haven't done it in a while, but incubating in a cold room overnight while a mixture of antibody rocks is not my idea of a good time. Zirconium and vinegar? Sign me up (plus the antibody is expensive and I've heard hasn't improved much in the last dozen years). Okay, on a re-read I think I'm not getting this right. Maybe it is only enriching the sulfo-Y. In which case I think I'll just study sulfo-Ys next time. 

Cool - so now they have peptides with modified tyrosines - how do you tell a 0.01something Da mod apart? On my instruments you don't. Particularly on a week like this one where we don't have heat in our building. My TOFs are like +/- 0.03something, though they seem a little better between 1 and 4pm. 

At very low collision energies, they neutral loss different! They use an NCE of 10 and that is enough to neutral loss sulfoY but not phosphoY! Then they can fragment it again with HCD or ETD or ETHCD or whatever. 

End result? MASSIVE improvement in our understanding of what proteins get Y sulfo-ed! What a cool resource for people out there who are into sulfo- metabolism and have (rightly) thought that mass spectrometrists can't really help them!

Wednesday, November 8, 2023

QuantumESSI writes least biased app note in history - bye bye LCMS, Ploopinum time!


This week I was thrilled to find that 8 new emails from newcomer QuantumSi informing me about their head to head battle with LCMS.

I was even happier to find out I had to go to their site and fill out a bunch of stuff about myself in order to access this app note. 

Possibly the greatest moment of my life was reading through what appears to be a high school student's interpretation of the experiments that were performed. It wouldn't have been at the pinnacle of my life's experiences without the requirement to agree to be Added. To. The. Mailing. List. to download it when I clearly followed one of the 8 links handily provided to my inbox because - presumably - I'm on the mailing list....

9th time's the charm, right?

Here are the details on the mass spectrometry

I don't know who did this for them, but - bravo - 5 day turnaround - including however long it took them to ship it to you? 

The Ploopinum system takes about 4 days including digestion, the 10 hours the sample spent on the instrument in their lab, uploading the data to the secure cloud and - presumably - doing cloud things on this single purified and digested protein.

But it outputs prettier data than the core did! Look at this! 

Just about every LCMS core has different levels of data that you can provide to collaborators/clients right? The phenomenal core at Pitt has "data processing tiers" and I think that is a smart idea. Some of my collaborators want to install the Proteome Discoverer viewer on their PCs and actually look at their data. Others, however, want a protein list like the one at the top. I'm lying, of course, I always offer the viewer software and processed data and in 3 years at this university no one has wanted it. They want the spreadsheet. 

Takeaway number 2 ? The Ploopinum isn't fast. Even if you have one in your own lab, for your purchasing of this box, the reagents and the cloud subscription service and the dedicated operator to do the experiment, you're slightly faster than an LCMS core service.  (Takeaway #1 is that this company's marketing could use some work).

Okay - but why is the Ploopinum so superior? That's the whole reason you're reading this, right? 

The Quantum device identified 2 peptides that the LCMS analysis did not. 

The first was a 5 amino acid section RLYCK (this was a LysC digested protein) because the operators only considered a 6 amino acid or longer region to be worth reporting to the end user.

You could argue, I guess, that the reason we ignore chains of 5 amino acids is that they are generally quite likely to occur at random throughout the proteome of anything - like, for example, the amino acid combination RLY is found in 11 proteins in the cRAP database. So....I ain't reporting that I found an RLY peptide to anyone. 

RLYCK occurs in 4 human proteins in the tiny canonical (no isoform, no common population level genetic variants) UniProt/SwissProt database. (It occurs 14 times if you consider common human single amino acid variants deposited as of the last time I updated my library in 2020). UniProt matches shown below. 

So...if this was a "proteomics" service request, most facilities won't report this peptide. 

And the Ploopinum identified a peptide with a pyroglutamic acid in it that the LCMS core did not.

Of course, the LCMS core was able to identify that peptide when they were asked to look for it and you could argue that it seems a little suspicious that the core appears to have been provided a purified protein and was not asked to look for PTMs in the sequence - like...maybe this went to a proteomics core and not to a facility that specializes in single protein analysis? ....but it's too late. The damage has been done. They handed this data to Billy and he already got a C on his writeup for his 9th grade general science research paper.  Someone in this company who wears a suit every day said "looks great Billy....let's not put your name on it, though, and the best way to get this marketed to the right people would be to send it to people with LCMS systems!" 

It worked! I've already powered down my mass specs that weren't destroyed in our most recent flood, because clearly this little instrument that takes 10 hours to identify 8 LysC digested peptides from a purified protein is the future. (OMG. Because some people can't tell when I'm being saracastic, no I'm not powering down any system here. On my oldest system -- that is no longer even manufactured - an S-Trap digestion of this purified protein would be about 2 hours (about 12 minutes of work) and I'd run this on a 20 minute LCMS gradient. I'd run it through single protein analysis software - NOT proteomics software - and I bet you I'd have better coverage of this protein by the time I'd forget to eat lunch today. But not everyone has this stuff around. And you can't get a degree in proteomics. You have to learn it on the job. 

Now - I've been pretty negative about this, but I tell you what, there is at least one of these things on the way to my university now. Because this little box has a lot of potential for researchers with a lot of money and without a lot of patience for me and my whining that my I need consistent temperature, relatively consistent humidity and dry floors in my lab to operate my mass spectrometers. There is a market for this little box. Despite the 9 emails I'll get every time they write up every app note, it isn't marketed for me, but it certainly will find homes in labs out there in the world.