LCMSMethods.org is back(?) again, on Protocols.io!

 

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 Protocols.io 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 Protocols.io 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 lcmsmethods@gmail.com

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! 

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. 

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. 

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.

OpenWorm.org here! 

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 community...you're welcome!) 

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! 

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! 

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! 

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.  

....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!