Sunday, October 31, 2021 is back(?) again, on!


I've got a lot of started projects and a lot less finished ones, with a dwindling level of energy and free time so I've had to pick and choose what to keep going. I honestly thought that this one was going to be less relevant now with vendors providing good methods built into their boxes. On the Old Timey Proteomics Radio Hour, I was surprised to learn that the vendor provided methods weren't real...popular...effective...? So maybe this is worth some time again?  Huge shoutout to Sierra Miller for bringing to the group's attention. 

There is a learning curve here (my first posted method totally didn't work), but two uploads are live. Go to and look up LCMSMethods. You'll find the old methods upload from 2 years ago that has 100-ish methods for the Orbitrap systems. You'll also find the first new method! How to do crosslinking (optimized for PhoX). This was contributed by Johannes Hevler who is doing his PhD with Albert Heck and Richard Scheltema, so I'm going to go out on a limb and say that this is probably a good starting method. I honestly had zero idea how to start with crosslinking on a TIMSTOF, so I figured this was a great method to post first. There are 2 uploads for this method, as I did the first one wrong (learning curve). 

If you've got methods to contribute, send them over to

If you'd like to help with this project (thank you thank you thank you) I would happily add you as a collaborator so you can upload and curate methods. 

This site is super cool because if you add a method you can create permanent DOI for that method and you can link that in your papers. Then everyone can just click that DOI in your paper and get your method and do what you did! 

Friday, October 29, 2021

Single cell proteomic method paper barrage! Last one for a bit.

 We finally have a nuts-and-bolts SCOPE protocol

100% recommended. My suggestion is to largely ignore the method sections from the first couple of papers on the topic. Things improved markedly up to SCOPE2. Still, stuff to learn in the first papers, but don't get the methods mixed up in your head and this is the one to tack on the wall. 

The automated NanoPots paper (NanoPots on a CellenOne, which makes it seem a lot less miserable of a process! It actually seems doable, if you've got $380k for a sample handling robot. Man, do people make funny noises when I bring that part up....) is out! 

The super cool R package from Laurent Gatto's lab with tutorials for how to work through single cell proteomics Orbitrap data is also out. 

Thursday, October 28, 2021

Fluorescent based sorting directly coupled to single cell proteomics!

I'd read this a while back and thought I'd posted on it and found two drafts that I'd started. TOTALLY worth checking out. 

If your are getting started in single cell proteomics (or want to), I strongly recommend that you watch some Youtube videos on cell sorting. I started out with some major misconceptions because I didn't actually know what the core that we use was doing.

I really like this video because, it's 8 minutes, hits all the key points and the references are directly linked below the video and you can read along as the work is being interpreted. 

Wednesday, October 27, 2021

OpenWorm -- A huge project to build the first complete digital organism!


I got bored with mass spec stuff for a bit. Brain break before some conference. I actually read some weird economic papers today, used some of that for the conclusions section of a preprint I really like and need to submit, but then ran into OPENWORM.

Github here. here

Tuesday, October 26, 2021

Your first in-person conference in a while? It's okay to not be cool.


Hey! So I don't know who needs to hear this (hopefully no one) but I feel like ASMS is a lot of people's first in-person conference in a long time. 

As what can only be described as the paradigm of mental stability in the proteomics community, I'm probably not the person who should be typing this, but I'm going to anyway.

If you're super fucking freaked out about a your first in-person meeting in a while, that's totally okay. 

If you're super cool and not freaked out at all about your first in-person meeting that's also totally okay. More power to you, yo! 

We're a small, but rapidly growing, community of weirdos who have some really odd common ground.

We're the minority of scientists who think that proteins and metabolites (and lipids and glycoRNAs, etc.,) are what really lets us know what is happening in a biological system. It is not even close to common knowledge that mRNA abundance doesn't quantify with protein abundance in mammalian systems. We're the outsiders who know this.  

We're the minority of scientists who know how to convert biological molecules to goddamned charged gases and move them around in vacuum chambers to not only identify them but accurately quantify them. Ever explained what you do to a taxi driver? You should. I have. Maybe I can't shut up, OR what we do is fucking bad ass. 

We spend a lot of time quabbling about who named what first and who's dumb p/q/r/s/t/z value is the best method for identfying something that we have 14 more points of evidence that thing is what we say it is than literally any other scientist who measures anything else on this planet in any other way,

but weird stuff like that also makes us a community. (It's still weird, though, most of those other assholes are using rabbit blood to quantify stuff and reviewer #2 in our field is like "where is your y6 ion in this 24 amino acid sequence, if I consider every possibility of amino acid structure that could have occurred by chance in the course of protein biology in the history of our planet there iiiiiiis a second possibility...)

All rambling aside, we're in the middle of a global pandemic that isn't over. ASMS is doing a stellar job of making sure that we're safe, but its both cool and natural and legit fucking okay if you're stressed out.

If you need time to yourself, take it. 
If you need to bow out of something where you don't feel comfortable, do it. 

If you're an analytical chemist (weirdo) and you start justifying the viral carrier load off an estimation of the total volume of air capacity in a given space to make yourself feel better about being somewhere, maybe you should just take a break. It'll be easy for peer pressure to feel like a thing. We all have our own capacities for exposure, risk and stress.

I was going to go with a title of "it's okay to not be okay" but apparently that's a soap opera in South Korea or something, but that's what I wanted to write. The world today isn't normal. Take care of yourself, and I bet you this little community of weirdos will have your back. 

Joke as required by blog rules after this many serious statements: 
And if you're totally cool you can always use the excuse that you're not to ditch a mass spec sales rep with your bar tab. Always works, and, come on, y'all have to have some sort of guess what the margins are like on one of these boxes. They're a solid 70% profit. If someone is selling a mass spec, they can cover a few shots of Patron. (To the sales reps in our're welcome!) 

Monday, October 25, 2021

Vendors are getting creative -- new mass specs are driving your way!


SCIEX is bringing some crazy new technology out to play and to get people exposed (to the technology and not to viruses!), I guess they're driving a truck full of mass specs across the country so we can see them outside in big open areas! 

In Baltimore, ours is stopping at the awesome Sagamore distillery downtown and there is a day of seminars (and food trucks) and people who register early get a distillery tour and get to take a drink on a boat!

Hey, stupid virus, try getting me through these 3 vaccine shotsy around my mask while I'm surrounded on all sides by the competitive fresh air of the scenic Chesapeake ba blowing off of America's greatest city. 

You can check here to see if your city is a stop for this truck and register! 

Sunday, October 24, 2021

PRM-LIVE -- Real time RT alignment for targeting on TIMSTOFs!


The TIMSTOFs are still relatively new devices and, while there are more of them all over the place all the time. My school has purchased 3 TIMSTOFs in the last 12 months! That's more than the number of Orbitraps purchased here in the last 3 years combined! There is a lot of enthusiasm about this hardware, but it's got some catching up to do. 

It's easy to forget how much of the stuff that an Eclipse can do was developed through external collaborations, or entirely externally. 

ETD -- UVA/Wisconsin

UVPD -- somewhere in Texas? Wisconsin too maybe? 

PRM -- I think someone in Wisconsin just gave it a cool name, but it might have come from there as well. I forget.

TMT MS3 -- Definitely Harvard

Lockmass -- I forget, but I think that was a Munich idea first

Real time search -- Harvard? University of Washington? I think it was a collaboration between those two places 

Internal Standard Triggered PRM -- I think one of these people had something to do with it! (This is a paper that I think of first out of Luxembourg, but I think because it was the first Q Exactive one)

In this new study, this group provides a 1 page python script that allows your TIMSTOF to adjust with your retention time drift! What did you say? Oh, your instrument can do that? Cool, well mine couldn't until this group stuffed a 1 page python script as the supplemental of this paper! 

I haven't attempted to set up TimeWarp yet, because the instrument is in use, but it looks straight-forward. This isn't all that you'll need to use it. You'll need the Python API which you can get by contacting your sales rep. It will require that you use TIMS Control (if you're using the older one that has a lot more features you can change, you'll have to switch over). If you haven't upgraded TIMS Control in a while you might need to get a newer one installed. 

In the bottom corner of TIMS Control if you see something about the Python API, but right now it's probably just greyed out. If you actually have something in the correct subfolder of your drive then it will no longer be greyed out and you can actually use it. Hopefully this is just the beginning! And if something this powerful can put together in just 575 lines, the sky might be the limit! 

Saturday, October 23, 2021

NanoScape -- You can now expense that VR headset you've been wanting!


Big shoutout to Richard for the heads up on social media on this one! 

Want to wander around in a cancer cell like you're in a video game? Hell yes you do, nerd! You can get it on Steam and it appears to be free! Importantly, there is a VR version. I've got to use my little brother's VR setup a couple of times and the Oculus is amazing, and stupid expensive. But...yo...this is for science now! If your accountant has a problem with it, send her/him this blog post!  (...actually...maybe don't do that....meh...whatever....)

The system requirements are kind of crazy. You need a minimum of a 1080 (which is still a great crypto miner, so the prices are stupid on them) and a serious processor. You might need to write off a PC upgrade.... 

Edit: I forgot to link the paper! Here it is.

Edit 2: I don't have VR so I downloaded the regular one on a PC I built 4 or 5 years ago that has much lower specs than recommended on the Steam page. It might stutter just a little as I'm walking around looking at embedded membrane proteins. It's cool that you don't need a crazy powerful GPU to at least run it even if it might work better with the extra firepower! 

Friday, October 22, 2021

Single quad in-source fragmentation targeted proteomics!


Wooo! I knew this was coming, and am excited to see it out

Some people in California have been investigating whether we need all this fancy hardware for targeted analysis. They started with metabolites, but - hey - why not extend it to peptides? 

It all comes down to confidence metrics, right? Is this your molecule of interest? How many points do you need to prove it? 

Some people go all the way one direction where they have:

Molecule retention time

Exact mass (or mass out to 3 decimal places)

Exact mass of every fragment ion (or out to 2 or 3 decimal places)

Increasingly, they also have ion mobility values of some kind (I'd go so far as say that FAIMS should count here)

And maybe they even have internal standards 

This is obviously superb and amazing and extremely high confidence AND ABSURDLY EXPENSIVE. Good for you if you've got the cash, but I'm in the U.S. and we need to make sure that extremely wealthy people get paid every time we need a medical test. 

If we want proteomics in the clinic, we need to cut costs all over the place. Years ago I was told $8 needs to be the target for an assay that can be billed to a patient for $800. Those costs have ballooned out, but it's tough to get there when you start with a $1 million dollar instrument.

Start with a $60k single quad? Now you can start talking about new assays that will make enough profit for hospital administrators and insurance companies that they'll let you even try to help patients.

The specificity in these matrices is suprisingly good. I highly recommend this study.  

Thursday, October 21, 2021

....and Coon Lab did the same thing with TEM...?


Huge shoutout to the Old Timey Proteomics Radio Hour for helping me get these two amazing new studies straight in my head and for the overall great discussions! There were 21 people on this one! 

Following up on the Cryo-EM deposition by mass spectrometry study on the blog earlier this week, here is a second (actually came out first, I think) study where a mass spectrometer is used to prep native proteins for microscopy

If you missed the Radio Hour, Jordan has been keeping notes and providing highlights. Here is this most recent one

Wednesday, October 20, 2021

Ever wanted to shoot native proteins out of your instrument and into a Cryo-EM?


I'm just going to leave this brilliant bit of insanity here

A Q Exactive UHMR (m/z range up to 80,000!) was modified a little bit so that native proteins could be cleaned up and deposited onto transmission electron microscopy grids for cryo electron microscopy! 

So....the biggest challenge with Cryo-EM is often QC'ing your samples, to the point that people will buy a couple QE UHMRs just to save some time getting good proteins....but then they can embed poorly or crystalize poory and ruin everything. What if you cut all the middle right out? Crazy. 

Tuesday, October 19, 2021

It's over. SOMASCAN crushes mass spec proteomics!


We all knew this was coming, right? Well, here it is.

Doesn't sound like the title that is the end of mass spectrometry? Well, they understated it. Pull down supplemental table 2. 

Turns out that SOMASCAN is WAY better than an Orbitrap Elite running TMT 6-plex reagent on human plasma. You heard that right. 2013's best technology ran well (the data was actually acquired in 2013) on one of the most difficult matrices, can't quantify as many proteins as SOMASCAN.

 How's the quan compare? The SOMASCAN is still better according to all this expertly crafted manuscript.

Wait. It looks like I wrote this, but I know I didn't because I can't follow any of this math. 

Okay, so the LCMS data is from an instrument that you can pick up at ReUzeit with a warranty for around $50k, so what? (I still love the Orbitrap Elite and would definitely take a free one if you have one you'd like to get rid of. Awesome frustrating to calibrate monsters that they are). 

They also correlate their SOMASCAN data with GWAS, which is a condemnation completely of it's own. Look, I'm not saying that SOMASCAN doesn't work, but if I was gonna try to prove a technology worked, I probably wouldn't go digging for the oldest proteomics files on MASSIVE and try to correlate my protein measurements with some 23andme data. 

Update 4/2/2022 -- Ummm....uhoh....okay, I recently learned that several of the top hits for SOMASCAN link to this blog despite the millions of investor dollars the company dumps into buying ad space. 
And that makes me afraid that a lot of traffic coming here is not going to catch the problems mentioned above. 

For a more thorough breakdown, I recommend you check out this post

However, just to summarize this post, I really put that title up to annoy a lot of other scientists. I honestly tell graduate students in our lab that they have my permission to ignore anything done in proteomics before 2017. There are exceptions of course, there was some great work, but the instruments were so slow that thorough experimental designs were very difficult to pull off. A lot of the problems in proteomics can be attributed to the lack of technical and biological replicates, but each sample today can take you between 10 minutes and a couple of hours. When I first moved into proteomics, the same coverage (at lower quality) would take days or weeks. If I were to compare my preferred hardware to another lab's hardware, I wouldn't be making a very good point if I put our newest instrument to their oldest, and I feel like this is what happened here. 

Sunday, October 17, 2021

Metabolomic links between depression and constipation in rats!


Science has been getting a lot of criticism recently, is the earth really round, does this SARS-CoV-2 thing actually exist, etc., What we need is a truly unifying study that everyone around the world can agree is something that needs answered and I'm excited to see that someone finally found the funding to explore: 

When a rat is constipated, is it depressed? When it is depressed, is it sometimes constipated? Does one cause the other? Is there a relationship at all? 

This team used NMR based metabolomics to explore these pivotal questions. 

To induce constipation, rats were fed white vinegar and activated carbon. It is important to note that this did not make them depressed, just constipated. 

For the depression model, the authors used a well-characterized system called the chronic unpredictable mild stress model. Just in case you aren't familiar here is a summary from the methods section of this study and the author's minor alterations: 

Which are, obviously, similar to the normal causes of depression in most mammals. 

What's the overlap? How do the two things relate to one another? Look, I'm not going to ruin this for you. You'll have to check it out for yourself here!

Saturday, October 16, 2021

Proteome Dynamics of bacterial sporulation!


Sporulating bacteria are responsible for all sorts of diseases. The little spores are dehydrated, surrounded by ridiculous numbers of layers of bacterial cell wall and other protective mechanisms that can make them resistant to extreme conditions. Some bacterial spores have been shown to resume vegetative growth after remaining dormant for thousands of years. Despite this, there is a lot we don't know about them.

Time to break out some new toys and do proteomics on them again, right? 

In this case, this group used a TIMSTOF Pro and 55 minute gradient, which probably seems like overkill for a bacillus that only has 3,000 genes, but hey, it's just an hour per run! 

What did they get for this? Well, this paper is extremely short and just kind of shows that proteomics can support 50 years of molecular biology used to figure out this model organism (B. subtilis), but all the files are on ProteomeXchange here

Thursday, October 14, 2021

Very different proteomic response to Vitamin C in male and female mice!


This new study ASAP at ACS's Proteomics journal throws me because of how very different these profiles are. 

Obviously, we need to be thinking about the effects that gender and genetics have (related point out of our lab)...

...and we all know this, but do we really know it? 

This group did expert level discovery serum proteomics on an Orbitrap Fusion, I forget which one now, and then they did expert level PRMs (maybe because they also couldn't believe how different these results were)? They spiked in the Biognosys iRTs for controls and I can't find a fault with their methods or data at first glance, and the serum proteomic response to something I consider as benign as Vitamin frickin' C in their models is very different between male and female mice. This really drives home for me how much we've got to think harder about proteomics in the context of biology and expand our matrices to get it all in. 

Wednesday, October 13, 2021

The mystery behind a 1,000 year old paintjob solved with proteomics!

Oh. This is too cool, and not the paper I meant to blog about this morning. It clearly says "top down thio-TMT one" on the sticky note on this monitor, but I found this while looking for it. (Top down TMT is super smart, though, and maybe I'll get to it) 

How do you paint something so that it stays red for 1,000 years? You start with mercury sulfide and someone solved that mystery 30 years ago, but there was something organic that held it all together and that was a mystery. 

Enter proteomics! With a tiny tiny tiny scraping of it and an Orbitrap Elite system this group shows that it was probably chicken egg and human blood. 

My favorite/least favorite part of this awesome study? 

Archaeologists have lots more things they want to run proteomics on, but it's too expensive??? For one, you can get an Orbitrap Elite like this one for $60,000 (often with a warranty) from ReUzeIt or ConquerSci or other second party vendors. 

However, if you're an archaeologist who has something awesome to do proteomics on I guarantee you we can find someone out there in the proteomics world who has time open on an instrument to squeeze something in. That goes for most things. Even core lab instruments have downtime here and there on Sunday nights. Network, yo! 

Tuesday, October 12, 2021

The N-Glycoproteome of K562!

 As you are dutifully moving away from the last experiment you'll ever do with HeLa cells or protein digests and starting anew with superior standards that are not unethical to use or sell, my first recommendation is the Promega K562 digest. (I 100% side with the family, and that company was warned this would happen.)

I've spoken to Promega about how they generate it and how they test for these scary mycoplasma things (thoroughly) and after running it at least once every day for QA on our TIMSTOF with extremely reproducible results (our daily QA is: 30 min LC gradient, 15 cm PepSep Reprosil C-18 1.7um with 3cm trap of same, "if 200 ng of K562 is less than 3,300 protein groups on FragPipe QC workflow, stop, something is wrong", which is altogether kind of insane, but when the system is freshly clean we come close to 4,000)

If you're thinking: "wait, is it as complex as that other cell line?" Check this out!  

5,000 N-linked glycopeptides?! That should be enough to QC your glycoproteomics workflow! 

As another option, NIST developed a liver homogenate lysate for proteomics. I don't think it's shipping yet, but it should go live soon. 

Monday, October 11, 2021

Great Clinical Proteomics Talk this Wednesday!


Interested in getting these mass spec toys into the clinic to help patients? 

Chris Shurford is this year's winner of the Michael S. Bereman Award for Innovative Clinical Proteomics. He's giving a talk on his work this Wednesday.

Sunday, October 10, 2021

Proteomes ARE proteoforms!


In all the excitement of "next gen proteomics" (i.e., the technologies that will soon make peptide quan by mass spec seem like a great way to waste both money and time while getting largely inferior data), mass spectrometrists will have several avenues to explore before those that won't adapt in time will end up relocating their labs to homeless shelters. 

This review in Proteomes discussed one such avenue nicely provided by evolution: 

Proteoforms! The basic fact that protein quan, while often better than trascript quan, is largely kind of dumb from a biological stand point.

Very few systems (at least in humans) are actually governed by a cell that made a whole bunch more of a protein. Sure, some of them do, but really that's not how any of these systems really work.

It's more like: kinase A phosphorylates target B and C. Target B translocates to the ER where it splices protein D. Phosphorylated targed C and spliced protein D form a tetramer with 2 of each of them and that tetramer migrates to the membrane and opens a pore. Most of the time 10 other things are ubiquitylated/ubiquitinated/sumoylated and THOSE are the actual phenotypes! 

When that new guy shows up at your university with that benchtop thing that can quantify 7,000 proteins in 4,000 samples before lunch, that's your survival plan. Move toward quantitative PTMs and start saying the word "proteoform" all the time, cause that's the currency we need to be using. 

I really like the paper above

I also think this is worth a read

Saturday, October 9, 2021

Single cell proteomics (and preprints) take center stage!


Chances are you've probably seen this picture of Ying Zhu and this feature in Nature a couple of weeks ago. If you haven't it is a solid read. Obviously, I'm biased, because basically all the plans I have for something called "tenor" involve applying single cell proteomics to better understand how drugs work. 

While this is a great read and highlights papers that we've talked about a lot in lab (with the exception of yesterday's ("yesterday"'s) post which I missed somehow. 

The thing that really jumps out to me is this -- can you imagine even 3 years ago, those old fogies (is that a word?) at Nature running a feature on 15 studies (whoops, they added one they missed) 16, studies where 7 of them were preprints? 

I think this is really important because preprints are definitely at a crossroads. 

The data is coming in and it's pretty conclusive: preprints are not getting cited in peer reviewed literature. Now, if a preprint is there and it gets accepted, it appears that having preprinted the study does help. Honestly, I think this may just be the fact that there are two places where the study will pop up in a Google search.  (Here is one reference, caution, this is the direct .pdf download). 

This is despite a completely scientific and unbiased survey that I performed in the Spring that came back with these results. 192 scientists is a pretty big number. There are only like 8 million in the world. 

A grant I applied for recently didn't consider preprints at all. They simply couldn't be uploaded or linked, so if you need grants, is this the best use of your time? 

Again, we're at some sort of a crossroad. What's best for the world? Obviously preprinting data, open sharing all of it and pushing to make the world a better place. What's good for individuals? Hopefully it will be those same things! This Nature feature suggests that maybe it can be, but it doesn't look like we're there quite yet.

Friday, October 8, 2021

Multiplexed DIA!!


This is from April?!?! How on earth did I miss this? 

Tiny window DIA on multiplexed samples!! My first thought is "of course that would work, but aren't instruments too slow?" 

I guess not, because this data (HF-X) looks pretty great. This does explain some questions I had regarding another preprint from this group where I was wondering if I missed something. Guess I did! 

Thursday, October 7, 2021

Is it really acetylated and where you think? Check the chromatography!


Acetylations can occur on multiple amino acids in your peptides and it's no secret that most search tools struggle with localization of modification sites (that's why we have A-score and ptmRS to help).

This group looked at loads of modified and acetylated peptides in terms of how they elute on reverse phase and other types of chromatography

If you're really stuck on that site and whether it's: A) real and B) where it is located, this might help? 

Wednesday, October 6, 2021

Immunopeptidoproteotranscriptogenomics illuminates the immunopeptidoproteotranscriptogenome!


This is a nice walkthrough on using transcriptomics to guide the discovery of new neo-antigens, including the use of synthetic peptides and MHC binding tools to help support your results. 

You might get to the flow chart on how they did the informatics and think about pursuing a different field of study, because....ouch....there are 40+ steps here. It sure seems simpler to not do the transcriptomics at all. I wonder when we'll hit that tipping point? Some journals don't require that you validate your mass spec data with western blots or other rabbit blood based techniques now. How far are we from just using the mass spectra and not needing the noisy crazy looking next gen sequencing data? 

In terms of the details, the samples were a leukemia monocyte cell line and the data was generated on an Orbitrap Fusion in high/high mode (orbi-orbi) with HCD. Most of the data analysis on the proteomics side was done in PEAKs. 

Tuesday, October 5, 2021

What's in a name -- picking reviewers, by Dr. Eyers!


Link here.

This is short and inciteful for anyone who, perhaps, just applied for their first ever academic grant and found during the process (at least through the system they used) you can't list preprinted work or even mention it, so maybe you look like an idiot for applying for something you can't list any proof that you can do. If you've been locked in your office working on getting papers accepted in journals so you look like less of an idiot when you ultimately reapply, good advice is nice to see and receive!