Now, More Than Ever, Proteomics Needs Better Chromatography!

 

Okay, I've definitely rambled about this before, but I learned from @DrJeanita (how have we not met before, I literally just go to NIST in Gaithersburg to hang out, and not just to lure people to the microbrewery across from the gate with me...but what happens pre-COVID, happens...if you don't know her, you should follow her) at the USHUPO mentoring day that it's okay to repeat really important stuff and this paper is SO SO SO important...

I'll go ahead and say it, CHROMATOGRAPHY....

And I've gotten away with pretty much ignoring it (for proteomics) my whole career. My QTrap got a decent MS/MS scan every second or so, my Orbi XL realistically got around 5 Scans/second, my Orbi Velos probably 8 scans/second and a QE Classic/Plus gets 10 or so. 

If you are only getting 10 scans/second....your peaks can look like the Appalachian mountains. The resolution of your chromatography isn't holding you back. You can probably only fragment the things at the very top anyway, unless your sample is very simple.

However, as this amazing paper describes in painstaking detail -- at some point your scan speed is no longer your limiting factor -- and many of today's instruments now are at that point. What IS your limiting factor? 

Ugh...as much as I hate to say it...it might absolutely be your chromatography.... We're seeing an increasing number of papers IN PROTEOMICS that are discussing chromatographic resolution, peak capacity and (...puked right on my keyboard...took a minute to replace it....) theoretical plate counts. 

I can't stay on this topic much longer without thinking hard about whether I was happier the 6 years of my life I spent washing dishes in the back of a bar-b-que joint in the south that didn't have air conditioning in the kitchn, so I'm going to leave you with a really staggeringly boring powerpoint from Agilent (direct link here) that shows how you can use Algebra to understand chromatography. 

If you're wondering why you can't get the results that the labs that write the papers that you hate you're collaborators carrying into your office are getting, at least part of it might be chromatography. Part of it might be that they're now famous enough that they can publish literally anything, but this sentence is probably a joke. We all know that's not how science works! 

The best free software for protein deconvolution!

Have I posted all this before? I feel like I have, but the question comes up all the time. 

Scenario -- you have a beautiful spectra of an intact protein like the thing above and you just want the deconvoluted mass and you don't want to spend $6k for one seat of software to get something like this?

This is the NIST mAB standard with 2 clear glycoforms (148,080 Da)

If you can pick out one single peak and get one pretty spectra like the top panel, there is a software from 1998 wrapped in a nice GUI that works in Windows 7 and 10 that will do this in under 10 seconds. 

This is the paper that explains it. 

And someone, not me, put up a link to the secret word-of-mouth, you can only get at ASMS from a friend of a friend if you bring a clean USB stick and meet behind the piano bar at a specific time.

The legality of the software is something everyone always seems worried about. Everyone made me concerned enough that I've never once put up a link for it, but since someone else put up a link with the intention of mentioning it to people a lot at the 2 overlapping conferences this week (ABRF & USHUPO simultaneously? Wut?) 

You can get it here.  There is an old video from some rambly nasaly person using an older version on Vimeo that eventually shows you how to use it. 

Okay -- so that works for extremely simple stuff. What if your protein isn't fantastic? You can go crazy sorting through and averaging MS/MS spectra.

Here is the question you need to ask next; Do you:

1) Want to see if your protein is there? 

OR

2) Do you want to look at the difference in the proteins between sample A and sample B? 

If I answer #1, then I go straight to UniDec. 

If you answered #2 then I fire up FlashLFQ, which I find easiest to use through the Proteoform Suite.  Proteoform suite is designed for big top-down, but I'd argue that this is just as good for antibody characterization as any of the expensive software packages out there. You need to convert your files to mz(X?)mL first and then you can deconvolute all the proteins in your file and do quantitative comparisons between files. 

Angry baby, gotta go. Hope this is helpful somehow! 

ProteaseGuru -- Take the guesswork out of picking your enzyme!

I just downloaded this and it's going on my desktop right now (if I ever close it).  

I feel confident saying that you need ProteaseGuru too! 

 

What is it? It's a really simple little program that will tell you what protease (trypsin, lysC, etc., etc., it has everything I can think of preloaded) is a good decision for your experiment and what you're trying to accomplish! 

There probably aren't too many proteins that people on this planet have spent more money studying than KRAS --

I highlighted the part that everyone I've ever met seems to care about. Trypsin ain't gonna do it. What will? 

You could sit there with a calculator and look at what the 7 different enzymes you have sitting around from past experiments might do -- or -- you could load your FASTA (with isoforms, etc, heck -- your ENTIRE ORGANISM FASTA) -- and just pick from an amazing array of digestion conditions 

(This thing is super easy to use, you don't need instructions if you're a lazy person) 

And -- we have a winner! AspN (with considering up to 2 missed cleavage) totally covers the whole lysine rich domain. 

Worth noting, there are other helpful tools here including handy histograms to visualize theoretical output. 

Don't feel like reading more than this? You can download this great piece of software at Github here. 

RawBeans -- Ultrafast, ultrasimple, ultrapowerful QC software for shotgun proteomics!

 

Once upon a time we had a cool program called RawMeat from VastScientific that was free and could rapidly look at any Thermo RAW file and provide all sorts of insight into your experiment. Vast got bought up by some corporation and all their software eventually disappeared (I'd still argue that peak finding software was competitive with anything out there right now -- it was designed for much smaller data files and can take months on today's massive data files). If you've got an older instrument RawMeat is still great and I can help you get it, but it doesn't work for anything beyond a certain Foundation version. 

I've mentioned RawBeans before, but wooo -- it's been updated and it's unbelievably easy and powerful now.  And it's got a paper now!

For real, it's super simple. You can get it here. And this is all you have to do (finding where Windows10 hides your MSConvert.exe is the hardest part). 

What can you do with a Lumos file in 101 seconds? (I have a weird mouse glitch but I ziptied all my PC wires together so they look really neat and I can't talk myself into replacing my infuriating mouse and redoing it all). 

(Click to expand, if you need to see the amazing output reports you can get). All of this populates into an HTML file. 

Did you get low IDs and you're wondering why? Run your file through RawBeans. If you maxed out your ion injection time then rerun the sample with a higher max ion injection time (also called fill time on other instruments). 

Best of all, it's vendor neutral! 

Tired of buying columns?!? -- pack hundreds a day yourself!

 

Packing your own columns might not be for everyone....

...but if you are someone who does this, you know that when you get that stupid slurry just right it sometimes makes sense to just keep making them until you have to stop. 

As an aside, I'm not sure if I've mentioned this, but due to COVID spacing we moved most of our instruments into Bob Cotter's lab space which has been used little over the years. One thing I found was the results of someone's great slurry day because there were around 20 packed columns taped to the wall! 

What if you had a great slurry day and you knocked out enough columns for yourself for the next 10 years? 

This preprint has all the plans to set yourself up to pack 50 columns in 100 minutes! 

Cranking up Thermal Proteome Profiling for Drug Mechanisms with TMTPro!

Thermal Proteome Profiling is one of the awesomely powerful new(-ish) methods for rapidly working out how a drug works in a cell. 

I've rambled about it here and there, but it was way too slow for me to suggest it to anyone around me until people started multiplexing it. (PISA blog post here that will link you to older posts/papers).

Basic idea -- proteins have a natural way of unfolding. If a drug is stuck to them or messing with them in some way, that natural way of unfolding is going to be disturbed. 

Why not get the whole experiment over with fast by using TMTPro? 

Interesting from a very boring level, the authors are using a Tribrid system and they don't change their fragmentation parameters for the TMT11-plex to the TMTPro. In every paper I've seen so far, people are using a little less energy, particularly for the Q Exactive family of instruments. There is some wiggle room in HCD for fragmentation on everything. At a purely anecdotal level, it has seemed to me that the HCD/IRM assembly on the Fusion isn't as particular about HCD energy as the terminal HCD on the Q Exactives. I can't back that up with any data, but at a big core where multiple users accessed each instrument, the Fusion HCD seemed less annoyed by users who used slightly different fragmentation settings. Maybe that plays in here?

Death to nanoLC -- 50uL/min proteomics, 38,000 samples and counting!

Hey you! 

What's your record for number of samples on that EasySpray column? Have you ever gotten 6 weeks out of one? I did once because I took the thing apart and put a new emitter in it after it clogged. (I should show you people how to do that! It doesn't work great, and it kind of negates the whole point of buying an EasySpray if you're going to make a crappy junction on the inside of one....but in an emergency.)

That plot above is the performance of proteomics columns with a time scale that isn't DAYS. It's over a 2 year period. Check it out here! 

How are they doing this? 50uL/min. 

The proteomics data quality doesn't suck, at all. 

The Lumos and HF-X systems with the big opening in the front do appear to be necessary to hit the kind of coverage this group does, particularly with the short gradient lengths employed.  In our hands we can get great data on the Q Exactive Classic, but we can't get away with 30 minute gradients and we have to use much larger injection volumes. (Preprint here I keep forgetting to submit somewhere so nice proteomics people can reject it.) 

High flow proteomics is coming and hopefully we'll see the Nanoflow HPLC start to die out. Your collaborators are prepping MILLIgrams of protein most of the time. You need nanoflow for nanograms of material. 

THERE IS FINALLY A PROTEOMICS PODCAST!!

 I don't know how to listen to a podcast, but I'm going to figure it out! 

You can check out the Proteomics old-time radio hour here! 

ABRF 2019 DIA Study Preprint is out!

 Are you one of the 45 labs that submitted data for the 2019 ABRF DIA study? Well, the results are out! And...if...you've got a spare hard drive and just want to see what this group of volunteers did....you can download the 1.7 TERABYTES of data acquired for the study at MASSIVE. 

Or you could check out the summary at BioRXIV here! 

On that topic, the virtual ABRF 2021 Abstract Deadline is 2/15. Which sounded far away until I was about to schedule this post to submit and my calendar stated that it is currently February of 2021. If I'd had to guess, it wouldn't have been my first one. So...coming up fast! 

Ethics in clinical proteomics!

Whoa! Almost 2 months without a blog post?  That might be the longest space in a decade, but we started a new project this year and he's kind of time consuming. (Thanks, Dr. Rinas for the great books!) 

If I'm writing the first post of the year, I figured it should be a really awful one. 

Let's talk about ethical principles and constraints in proteomics. Ugh. 

(Yes, I deliberately used the page that would have the Elfseverer logo on it, because that is the global stamp of ethical behavior.) 

This study is mostly a literature search about the stuff that we've gotten away without thinking about (ethics stuff) that the genetics people have to worry about. Wait. Why do we have to think about this all of a sudden? 

(Paper here)

This shouldn't come as a surprise to anyone, right? If you've got access to a mass spec and you haven't ran your own blood and some dog poo that looked funny through it, you're the weirdo, not me. Somewhere I've got slides from a lecture where I show that it looks like I'm lacking some important protein in my blood. In reality (from my whole genome sequencing data) I know I've got several single amino acid substitutions in that protein that subs in some Rs and the alternative cleavage events make them look downregulated since those variants aren't in uniprot. Impute the missing peptide and whoa, super downregulated. In this study they show how they can tell participants from past studies apart, and what info they can also extract on them. Fun! 

And...I guess if you're in a group where you've published more than one method section over the years with a statement like this....

....leading the charge on the ethical use of that information is a great idea! 

See...everyone does it! 

(Side note: This paper is quite old and I doubt with the coverage at the time that this is nearly as big of a deal, but I really thought that this was funny)