Thursday, August 31, 2023

5 years later -- where are we in proteomic biomarkers in body fluids?


I'm 90-ish percent sure that the last review is on this blog somewhere, but forget that one - check out this new one

Here's the thing -- the 2017 or 2018 one pointed out that proteomics was 

.....too slooooooooooooooooooowwww.... for what we needed for real biomarker discovery (big cohort size to encapsulate human variation).

Slow instruments, slow data processing, slow data integration with clinically revelant information. 

And you could take a step back and think that maybe this was written on a wall somewhere and this group spent a lot of time integrating every technique they could to start checking things off the wall. And where they didn't integrate, they created. 

Obviously not the only group really pushing things out from the discovery phase toward the clinic, I think Jenny's group at Cedars is on pace to double the number of LCMS based clinical assays that exist here in the US in the very near future.'ll see a lot of adoption of the same types of technology out there as you see reviewed in this study.

Now....the downer in this review is that we do honestly need to start thinking about ethical use of our data and how to ensure that when we're obtaining deeper understanding of our patient samples. It's only a matter of time before we'll absolutely have to start using encrypted storage servers for our data and instituting document storage/destruction protocols and it will be interesting to see who mandates that first. 

I'll stop here before this post gets longer than the review, but if you need some optimism injected in your day, it's short and open access and we've come a long way in 5 short years. And that's actually only 3 because COVID years don't count against us in any way. 

Wednesday, August 30, 2023

SCP Viz - A universal toolkit for single protein analysis in single cell proteomics data!


Hahahahahaha! Another self-serving post already? 

Not entirely, please read to the bottom! 

Okay -- this is something that I really desperately needed since I started doing single cell proteomics basically full time (around mentoring, grants, trying to keep the mass specs in my lab NOT UNDERWATER, trying to convince biologists that mass spectrometry COSTS MONEY EVEN WHEN WE COLLABORATE) and other fun things. 

And Ahmed Warshanna wrote it for me! Early print here.

Imagine this scenario -- you do a silencing RNA experiment or treat cells with a drug that should drop the abundance of a protein and you just want to figure out how well that actually dropped the protein abundance across a couple hundred single cells. Try visualizing that shit with anything you typically use. (Actually PD does a decent job until you get above like 800, but nothing else really does). I've made heatmaps using conditional formatting in Excel from the FragPipe and DIA-NN output and that sort of works and I can drop that report into GraphPad and wait a week for it to make what I want. I came up with a klugey use of relational databases through GlueViz in Anaconda (which I do honestly still use, but it's clear there is some facet of my particular neurochemistry that makes this compatible for me and even close friends trying to be supportive are really wondering about all that acetonitrile inhalation has done to me) and I don't think it has EVER been downloaded from the Github by anyone.

Introducing SCP Viz!  

SCP Viz lets you put in your SCP data from Proteome Discoverer or FragPipe or SpectroNaut (and - everything else, let us know if it doesn't, the preprint features scSeq data as well!) and put in that protein you "silenced" and visualize how it changed. Either the raw abundance/intensity from the software (or the normalized data if you had FragPipe MaxLFQ it or whatever) data across every single cell, or the box plots or histograms or the violin plots nerdy reviewers seem to like that I literally do not understand what they are showing at all. You can log transform your data or impute it and then you can use a sliding scale for your background imputation (I'd rather just see zeroes personally, but people do impute for whatever reason) but here is the coolest part.

Imagine that you've got 50 cells where you've still got background expression of your protein. Your "silencing" doesn't appear to have silenced anything or your inhibitor doesn't seem to have inhibited your protein. What makes those cells special? 

Use a box select or lasso select of the proteins exhibiting the WT or higher abundance (random protein shown, I gotta publish some things) then Unleash that Analysis (that was a compromise it used to say F' it) wait a minute till R does all the things in the background then download a file that only contains those special cells with that higher than normal protein abundance! 

Now you can import that CSV with your subpopulation of cells and really dig into what makes them special. Are those stupid things all twice the size (histone H4 signal is off the charts?) are they using a pore to pump out the silly RNA or drug? Now you can actually get to that data. I've been copying my headers across so then loading the whole text file back in with the important cells flagged so then I've got multiple populations to look at. 

Github is up and we actively (desperately) need users and collaborators. We found a funny glitch after submitting to biorxiv for TMT data we downloaded from the cool TMT cell death study and correcting that is in works. It's a living breathing document/program that will improve a lot as we test more data.

And here is why this post isn't entirely self-serving and why I pushed this out while we're still doing some bugfixes. 

Like just about any American kid who didn't luck into being born rich, the author of this software had to borrow a lot of money to go to college. They'll hand you tons of money to cover the spiraling out of control costs of higher education in my country (last I looked a year of undergrad at the school where I work was pretty darned close to what I take home per year....)

And thanks a corrupt pile of garbage in power, and especially this trash

everyone has to start paying their student loans back starting on September 1st. We can bail out corrupt bankers, forget to tax billionaires, spend trillions on bullshit wars to murder millions of people - as long as they're darker in complexion and far away -  but we can't help an entire generation of young people in this country. And the author of this software can't afford to work for me for the ridiculously low wages that I'm allowed to pay him out of my grants when he has to start paying his loans back. 

SO if you see an application for a young trainee in protein informatics cross your desk, do me a favor and give it a serious look even if it isn't the number of years of experience you were hoping for. Ain't the first paper with his name on it that you'll see in the very near future. 

Tuesday, August 29, 2023

10.18.2023 Proteomics Battle Royale Round 2! Mark your calendars!

As some of you are aware, at US HUPO in Chicago this March we decided to have a couple hours to cut through the crap and get into the technical details of some different proteomics technologies in the poorly named "LFQ Battle Royale". 

Despite technical issues like it being very hard to put a luchador mask on over glasses, we had this event. It was strange, it was funny and I took like 10 pages of notes. Unfortunately it was limited to the 12 or 150 people who could fit into this room in Chicago. I had trouble seeing because of said luchador mask. Who knows. 

US HUPO VMO will be hosting round 2 virtually so everyone everywhere can attend. AND THIS TIME we're really really cutting out the crap for the comparison everyone probably wanted.

Brought to you by the VMO - and you can check out our snazzy updated page here. 

Monday, August 28, 2023

US HUPO VMO Video competition! Get your cameras ready!



Official news and announcements are coming, but this year for US HUPO the VMO has successfully obtained funds to have a VIDEO COMPETITION. Please check on the US HUPO site to find the rules and stuff, I know we discussed them and read them all but it's like 6am and I can not be trusted with details. 

Basically - you get 3 minutes to film something about proteomics in general or about applying it. The goal is for us to sort of get out of our scary mean mass spectrometrist wizard caves for people and to attempt to be approachable.

VMO will review the videos to make sure they're appropriate (whatever that means) and then at US HUPO in Portland we'll show the videos and vote on them. Winning videos get money (for themselves) and bragging rights! 

I didn't know this, but apparently there are similar things out there. The chromatography crowd has been doing this for years and people have been "dancing their PhDs" for a long time as well. The former is relevant. The latter is surprisingly funny.

Sunday, August 27, 2023

Back online, I had to upgrade the blog to new Google rules.

Google Analytics kept sending me emails telling me there was a big deadline on 7/31 that required me to do things, but I have a lot of things "I urgently need to do" in my Inbox, and this doesn't involve patients or my staff so it comes last. locked me out.... While I was there I found this crazy chart. 

Probably just how Analytics tracks page views now? I doubt that blog traffic doubled in August vs. June when 3 cool new instruments were released. 

Imma pretend that twice as many people are Googling "proteomics" and surprised to find the top hit is something that makes very little sense.  

Proteomics to the moon! 

Saturday, August 26, 2023

How do different methods for enriching extracellular vesicles from human plasma compare?


A lot has been made recently (again) about the power of gaining proteomic depth (and biomarker discovery) in extracellular vesicles from plasma or serum or blood products (or other body fluids? seems likely). 

About time for an up-to-date review on the topic, right? Check this out! 

Thursday, August 24, 2023

MS Annika - Find all the crosslinks!


I've been using this for a while, and just saw the paper ASAP at JPR while seeing what I missed during a travel marathon.

You can get it at, but the instructions in this manuscript are super helpful if you're doing somethign harder than just MS2 of standard X-link reagents! 

Tuesday, August 22, 2023

ESPC Vienna! IMP is ready for precision medicine. Is your institution?

Holy shit, yo. Not to brag in a great big orange box where I type words, but this might be the best mass spec conference I've ever been to.

1) Vienna is AMAZING (that's my own bias)
2) I got to meet a bunch of people I've respected for years and get to sit down and talk to them finally. (Don't worry, I didn't do my whole "you're the bass player for In Flames, wanna see your band's logo on my butt" thing) Disclaimer. That is a joke and is NOT real. And there definitely isn't an Orbitrap tattooed on my upper leg (not my butt, that's different) either, I don't care who says they were there when Karl and I got them. 

I was legit cool, most of the time, but holy shit, this was a great meeting 

3) And I do not, in any way mean this to reflect poorly on any other meeting I've been to. 

European SCP was super cool, though. Obviously, we all have schedules, so we missed a couple of the international leaders in this space, but Europe is embracing single cell proteomics at a level that is unbelievable.

Right before I arrived the ESCP it was announced that my good friends at the world leading IMP Proteomics Facility had received an award to help support their goal of applying Single Cell Proteomics to help human diagnostics -- in the form of a $2.5M Euro award! 

And you might think "big deal, the rich got richer in the form of a bunch of cool stuff for a facility that has everything" and I really can see that, but you'd be surprised by what these facilities here in the middle of Vienna end up doing. SO MANY RESEARCHERS are supported her through this world class facility. Yes, there is a lot of world class talent, but then 



A SURGEON TELLS YOU THAT single cell proteomics (SINGLE CELL PROTEOMICS) HELPED THEM DIAGNOSE WHAT THAT TUMOR TYPE WAS-- that a pathologist completely mis-diagnosed using normal clinical means!

There was a surgeon right here. Right in front of me. Showing that single cell proteomics helped him diagnose a patient correctly! A real human being!  That could someone you love and this surgeon showed the surgery and thanked Peter and Karl and their labs for helping him with his work. And I had trouble talking for a while, because if you can tinker with mass spec toys and push out data good enough to help someone who cut a hole in someone's skull - that's something to be proud of for the rest of your life. 

And there was all sorts of amazing data here and amazing brilliant people and scary impressive young people doing shockingly good work, but IMP is digging in for the future of precision medicine and embracing single cell proteomics to help make it happen. Honestly, just an amazingly overwhelming couple of days and super cool meeting. 

(Edited to remove multiple bad words) 

Sunday, August 20, 2023

A review on recent trends in phosphoproteomics!


I missed this extremely thorough review from last year somehow and want to make sure that it's up here before I figure out which button on a complicated looking machine has the highest caffeine extraction efficiency. It covers just about every method I can think of. The review, I mean...

What a great review to kick of European Single Cell Proteomics 2023!

 I'm lost somewhere in Vienna, but my watch gives me credit for 3 hours of sleep on the plane and ESCP starts anytime now. Instead of making slides, I'm blogging, and I wrote a review that might not make any sense at all.

AND check out this great new review! 

Why would you read this after reading 11 of Slavov lab's 83 reviews on SCP? 

A chart with price per cell written out like a Yelp review! 

Saturday, August 19, 2023

Phospho-Analyst! The best phosphoproteomic analysis toolkit ever?


How good is Phospho-Analyst? It's break out a Randy Savage GIF for the first time in 4 years (don't check the accuracy of THAT statement, please).


Paper is here! 

Got whole proteomics data AND phosphoproteomics data? Leverage that! Is it basal level ERK2 expression and all of it is phosphorylated now? Or did your condition cause ERK2 levels to go through the roof and the phosphosite occupancy is unchanged? 

SNAP! Find out! 

Are you already using the amazing tools out of Monash like LFQ-Analyst and FragPipe Analyst? If not, you should be. They're AWESOME. If you are -- you don't even have to read. You already know how to load data and get your beautiful plots and find the sense in your data. 

You can access Phospho-Analyst here! 

Friday, August 18, 2023

Single cell proteomics for everyone with desktop single cell isolation!


Hmmm.....let's write this one carefully.... 

Y'all, it is really great if you can afford a $400,000 robot to very slowly isolate and prepare your single cells. The data consistency is amazing. It is so so so good. But you don't HAVE to have it unless you absolutely will only be getting 800 cells and you need to analyze all of them. Then there is nothing better. It'll put those 800 cells where you want them. 

You can do what I do and sit down with the people who run funny FACs things and get to some common language. They're just like us in many regards. Some asshole in a suit will walk into the university and tell the leadership there that if they spend XX million dollars on this big new thing, then they could get any dude shooting up fentanyl on their commute into the car to put on gloves and run the thing for 7 fentanyls an hour. Who needs an expert? Sound familiar? 

OR. There is this thing. Benchtop and "~1 order of magnitude" less expensive than what you were considering buying.

NanoDESI on a TIMSTOF - did this just lowkey show the future of spatial proteomics?

Okay, so you might think when flipping through this something like "big deal, some mass spectrometrists in the midwest stuck a klugey looking DESI source on the front of a different mass spec. Mass spectrometrists do all sorts of weird things"

And I'd agree. Until I got to Supplemental S5. 


You know what you can NOT do right now on an imaging TIMSTOF? You can't use that PASEF thing. You know...the thing that accelerates the instrument by up to 300x and increases the sensitivity by 100-ish fold? It don't work.

Don't get me wrong, this study is super cool. They get a resolution of 300 IMS and some sick lipid imaging across the board, but I think it lowkey showed that you could have your imaging resolution AND ridiculous ion mobility AND modern mass spectrometry on the back end. 

Of course, I might be dumb and they just targeted those lipids. OR this just sneakily showed what the future of spatial proteomics might be just around the corner. 

Thursday, August 17, 2023

QuantMS - Has proteomics (re)discovered the Cloud?


I often make this joke that if it looks like I'll go a month without getting a paper accepted, I'll just find the nicest dataset that one of you have put out here and I'll reprocess it on a computer with more than 4 cores using something called "the Cloud", find what you missed, and - violin! - new paper! If you haven't heard of "the Cloud" chances are you're in proteomics. "The Cloud" is when you log into someone else's huge ass computer and use loads and loads of resources for a short period of time. Then you can do crazy stuff like process a file in 10 seconds - or 5 minutes, if you want to do something stupid like take the time to look for 300 PTMs and using a database of every known human genetic variant.  

Sure screws up my plans (and the plans of 10 recent big $$ startups that have basically the business model of "searching proteomics data with 'The Cloud' to actually see what is in those files) if y'all all start using real computers.... 



When do you ducking sleep, yo? It's like trying to keep up with James Fulcher or Amanda "35 published papers before I got my PhD" Smythers. Absurd level of productivity out of people who aren't anywhere near as old as the clip I made that meme from....

Anyway, check out the Cloud thing above. If anything, it'll give me less reasons to make fun of our community to outsiders.

If you can't wait, or if that requires you to know code, the way I use the Cloud is to pay $20/file (might be my academic discount and I pay for 150 files at a time, so I might get a bulk discount) to OptysTech to use their Cloud computing platform, Bolt. You can pay extra for informatics support which gets you pretty cleaned up reports. I should put up an additional disclaimer (in addition to the ones over there ---> ) that the CEO of OptysTech is a long time friend of mine and we were awarded a grant from NCI that ran from 2018-2020. I'm just a paying customer these days, but that sounds like a conflict of made up blog rules to me. 

Wednesday, August 16, 2023

Single cell chemical proteomics investigates commitment to cell death!


How the actual duck did I miss this until just now?!!?!?  It's really really good and over a year old. If I saw it, I forgot. I was at the beach with my parents the week it came out and my Mom really hasn't recovered from how much my existence cut into her fun in high school. You don't...get...umm... smarter on vacation with my Mom. So maybe I read it and the brain cells didn't survive? I don't know, but it is super weird that I haven't been reading (AND CITING) this! 

I guess it is in Anal Chem and I don't really keep up with that. You never know when it's some dumb thing about silica binding, but I'll add that Zubarev guy to my Google Scholar alerts now. 

The data is all up on PRIDE here. Don't do anything with it until I download it, please. 

Here is why I'm super excited -- we're giving cells drugs that we want to kill them. Every drug I order I hope leaves a smoking crater in the cell culture hood where those cancer cells used to be. And I want to be able to have one of these poor mice eat 14 pounds of it and still have plenty of energy to bite me if I'm stupid enough to open that cage. However, it's hard to study a cell that has burst into flames (or finally given up and committed to programmed cell death or autophagy or kicked on 11 Caspases). 

This single cell chemical proteomics study very cleverly investigates the cells that are committing to death procedures. I can't wait to spend more time on it. 

Tuesday, August 15, 2023

Does Moore's law apply to mass spectrometers?


Moore's original law says something like the number of transistors on a chip will double every two years or something but it appears to apply to all sorts of other technology things.

How's it line up for mass spectrometers? Thats what this group looked at! 

There are a couple of punchlines in this study, but it's a 5 minute read, so you should check it out if you care. I'll give you the answer of - based on these parameters? Sorta. 

Monday, August 14, 2023

Making meaningful connections! A universal guide to NanoLC connections!


Wow wow wow. This blog post is a year old from MS Wil but if you're trying to do ANYTHING AT ALL CREATIVE with nanoLC plumbing you've probably gotten to the 


stage. I sure have! 

What you really need is to make a meaningful connection. And here you go! 

Honestly, I always hope we're at the point where I don't have to ever think about using bare fused silica for anything and....well....we're almost there if we really like Dr. Maitsch C-18 ReproSiL or PepMap. Outside of that.....this guide should help! 

Sunday, August 13, 2023

How much can RiboSeq add to your proteomics data? The definitive review!


This new review is SO good and if you happen to be reviewing a grant application with my name on it, please keep in mind that it wasn't out when we wrote said grant.

Does this help refine our thinking for multi-omics integration? Honestly, it doesn't change our plans but it does help really fine tune how to think about false discovery rates and true positives. 

AND is a really pretty review that brings up some real fundamental questions -- like -- "what really is a protein anyway.

100% recommended. 

Clear up the confusion and regenerate your productivity with FragPipe DIA!


A very common conversation I've had the last year is about the DIA-NN + FragPipe paper and how no one has been able to run DIA-NN successfully through FragPipe.

(this one) 

Around version 17 (I forget) a new DIA-NN tab appeared in support of that preprint and that sounded great, everyone tried it and no one could get it to work.

I even went to the amazing (how they possibly support it at that level) FragPipe Github support and asked the question and cleared it up for myself. 

And the confusion can be cleared up by this statement: 

You can't actually run DIA-NN through Fragpipe. You can quantify things using DIA-NN in FragPipe, but you can't identify things with DIA-NN in FragPipe. 

If you want to identify things AND quantify them, you need to use DIA-NN. Don't use Fragpipe.

Got it? Me too! It just took me a while! 

Let's clear up the confusion ever further! Forget DIA-NN entirely! Run MSFragger-DIA through MSFragger! 

What's Vadim doing on this paper? I dunno. I think we're all just supposed to put his name on everything now if you want a paper in a Nature family journal. Or he's collaborating with just about everyone. One of those things. (Edit. I'd just like to clarify that this is a joke, I think we're all floored by the output of this young researcher and the quality of work that he's consistently putting out there in the world.)

Why would you switch it up if you're already using DIA-NN? 

YOU CAN REGENERATE YOUR CAREER WITH TIME SAVINGS! Hard to tell from the scale, but is that 1,000x faster? 

And check out the UpSetR on the very top of the post in Figure A. The same results in 1% of the time? 

Saturday, August 12, 2023

Proteomic Analysis of Dracula (Vlad the Impaler!)'s letters!


What isn't to love about this amazing new study? 

1) I can FINALLY use this Symphony of the Night gif.

Wait. That probably shouldn't be number 1, but that is probably one of the best games ever made. 

Real #1) This team extracted peptides from actual letters sent by Vlad the Impaler - THE inspiration for Bram Stroker's ridiculously awesome first person account of meeting the fictional Dracula. 

Off topic, but if you've never actually read it, the fictional account of Dracula is letters from a lost-in-the-middle-of-nowhere nerd writing letters to someone back home as he ends up hanging out with the monster himself. This will email you the individual letters on the day they're written each year. Warning, he just whines about food for the first few weeks, maybe days, but it is hard to feel bad when very bad things happen to "the author". 

3) It is easy to think with these "proteomics unveils history" things that there is some level of extrapolation and human obsession with patterns, but - I dunno - this is a riveting story.

Is there blood on them? Fuck yeah there is, but that's just the beginning. 

Friday, August 11, 2023

Cutting edge INTRAMURAL proteomics at the NIH?! Check out this week's podcast!

Lazy post-tornado blog post while I've got internet and a minute to spare.

This week's episode of The Faces of US HUPO features Dr. Aleksandra Nita-Lazar. She's a young and dynamic proteomics researcher applying proteomics to understand how the human immune system reeeeaaallly works. 

You can check out her lab's website here. 

Now...Ben and I forgot to ask her anything about herself and we just asked questions about her work and how intramural research at the NIH works. So you'll just have to believe me that she is a fun and interesting person who is also doing very important work.