glycosylated RNA (glycoRNAs) and tools to help identify them in LCMS data!

 

I was kicking around the idea of submitting a formal review on this topic, but I'm not sure I have enough data for more than a really cool blog post. If you do take this and write up a paper feel free to acknowledge me. I won't object. 

If you haven't seen this paper, you should.  This study presents compelling evidence of an entirely new class of molecules that we had zero idea were even around. The ramifications of this are at the level of text-book-altering. Glycosylated short RNAs sticking on the surface of cells doing -- presumably -- super important things. The initial study only identifies a few and they appear to be annotated as: 

"Non coding RNA" -- which means, they don't appear to make proteins. Why are they there? No idea. Till now. (Kinda).

I just realized that picture above from the PDF cuts off 5 of the author names. Pedro, Benjamin, Alex, Benson and Karim deserve to have their names shown in the PDF. Get your act together, ElfSeverer. Fixed it. 

Without running this out forever, these authors did some really innovative labeling and RNA stuff that I'm sure makes sense to RNA people. And these cool molecules disappear when you treat with something that cleaves RNA or cleaves sugars. They also did some top notch LCMS work. You can find all their RNA data on public repositories, but I can't find the LCMS files. It was obviously something that was secondary to their goals. 

It is not, however, secondary to my goals. And I bet that there are a whole bunch of people weird enough to read a proteomics blog that don't know that there are great tools out there for looking at RNA data by LCMS. There are, in fact, even SEARCH ENGINES and a couple of them are in formats that you already know how to use. 

A couple of things real fast, though. 

1) Nucleotides don't like to ionize in positive mode. Neither do glycans. However at a certain size of a molecule you can pretty much stuff a proton on it somewhere, but the signal might not be great. If you haven't calibrated your instrument in negative mode since installation, you might be operating at a handicap. 

2) Nucleotides LOVE to fragment. No joke. Check this out. 

I highlighted it but it's from this amazing recent study 

See what I highlighted above?  You know what that means, right?!?!?

No, not popcorn time....geez.....it just rhymes....

What it means is that the tools are in the friendly, open, ultrapowerful, free...

OpenMS! I was using 2.4(?) for intact protein work and I had to upgrade to 2.5 in order to use the RNA sequencing tool that this team used in this study (NASE). Honestly, this is probably the best tool for the job today from an automated standpoint, but it definitely isn't the ONLY tool for the job. If you use OpenMS, you're set, though! 

My bias toward using Proteome Discoverer is well established by now. So the first thing I tried was messing with the settings in the OpenMS community nodes RNxPL! You can get those here. 

They're from an older study by the OpenMS team where they used UV to crosslink RNA to proteins and then tryptically digested them to figure out what RNA is interacting with what proteins. Super cool workflow that I have always kept in my back pocket for conversations with people interested in RNA-protein interactions. It's hard to see in the screenshot above, but there are these extra yellow/green lines. What are those? They're nucleotide diagnostic ions!! No joke, this tool is super cool and really easy to use, and it's got amazing visualization capabilities. My only issue with it at all is that the diagnostic ion mass accuracy is a little wide and I can't figure out how to change it.

Please note, it only works in PD 2.0 and PD 2.1. You can always go to the FlexNet and get an older version. They're easy to install and run. 

Want a stand-alone executable with a bunch of power? You should check out RAMM! The RNA Mod Mapper. It is one of many tools for studying RNA by mass spectrometry that you can get here.  You can go down a complete rabbit hole of nucleotide mass spec work from the Limbach group that will run you back to some of the biggest names in mass spec and chemistry that he's worked with over the years. He postdoc'ed with some guy at Utah who's name comes up in undergrad chemistry.  This site is an absolute gold mine. 

What if you just want to do some manual exploration? For example.....wait.....we did some weird shearing experiments of growing cells one time...right...?...I can't remember exactly why but we wanted stuff off the surface of cells without killing them. Still have those RAW files? Just curious if you see an MS/MS spectra with a HexNaC oxonium ion and a nucleotide fragment ion or 6? 

Or...just entirely new to RNAs and what they are or how they might fragment? ARIADNE time! 

Paper link! 

AMAZING Ariadne web resource! 

If you're using Xcalibur, I will warn you that it is a little frustrating looking for multiple diagnostic fragment ions. 

My recommendation is to make multiple overlapping ranges that are just filtered on MS2. (See below)

To do this, activate any MS2 spectra and then delete out the information. Then hit OKAY. It will then activate the filter that it doesn't want to that is only for MS2 spectra. Then you can put in your target ion masses. Once you have one done, you can highlight your first BasePeak (right below "Type") and then if you checkmark another box, then it will copy those settings. All you have to do is repeat and type in multiple diagnostic ion masses. In the example above, the 306.0491 fragment mass is pretty rare, the second ion (I think I used a HexNaC fragment) is much more common, but at 17.01 minutes I've got an MS/MS spectra with each within a 10ppm mass tolerance.

Is it a glycoRNA? No idea, but it might be worth trying copying that spectra out as an MGF and putting it into the RNA ModMapper! 

Ariadne isn't the only web based resource for modified RNA masses. It's just got the best color scheme. Another amazing resource is....

which you can access directly here! 

I can keep going, probably, but I should probably work on other stuff. 

One thing I'm currently hunting is SOS. If nothing else because the interface looks super cool. 

You can read about it here. 

See, I found a ton of cool stuff, but probably not enough for more than a fun 30 minute blog post. And this really does just look like the tip of an iceberg. 

OH! I almost forgot! 

Why am I even thinking about these to begin with? RNA is totally amenable to separation with C-18. Waters has a 90+ page application manual for studying oligonucleotides by LC and LCMS. They use a slightly different BEH C-18 column, and negative mode for most things. A direct download for the Waters PDF manual is here. 

What I'm thinking is that there are lots of situation where we may have already accidentally have acquired data on glycoRNAs or RNAglycans and they're just hanging around in the background of our data. How much of the "proteome dark matter" could be linked to this? A bunch? Who knows?  I don't use nucleases when I prep my shotgun proteomics samples.