Friday, November 27, 2020

The first TIMSTOF post. First week impressions!

I'm a die hard Orbitrap fan and I always will be. However, there are finally some other technologies out there that can compete in some ways with Dr. Makarov's incredible invention. I don't know if this has made the blog or not, it's been a busy year, but I've finally found a long-term position at JOHNS HOPKINS. I'm junior faculty in Namandje Bumpus's lab and we've got an amazing group of smart fast moving young people and a bunch of crazy ideas. Point of evidence? That ceiling disrupting monster in the picture above. We did a lot of research and compared a lot of data over the last year and a lab with all Thermo instruments filled most of a room with an instrument made by the NMR company? Sure did. That 2,000 pound 8'7" monster is the TIMSTOF Flex + MALDI 2. 

There will probably be a lot of TIMSTOF posts as I struggle through learning this thing and making it do what we want it to do (much of which it doesn't seem to want to do) because writing about it will help me think about it. Here are some very early impressions and some information I would have liked to have had going into the last week. 

First impressions. Installation: 

It's frickin' huge. 

The truck that came to deliver it, was TOO BIG for our loading docks. The 5 crates that arrived (2 of which were over 5 feet tall) does not include an HPLC (which, if you get from Bruker, would be box #6).  If you have any space limitation of any kind, you need to really plan this out. Boxes need to be opened in a specific order, because some crates are just the equipment necessary to move and install the rest. 

You'll also need to be prepared to remove lots of doors -- and to probably get on your neighbor's nerves for a day or two. Recommendation: Have at least 2 people around in addition to the FSE, and prepare for a solid 12 hours the first day to get moved and powered up. 

In general, though, the installation was pretty clean once the behemoth was in place. There is, of course, a federal law against any mass spectrometer manufacturer providing you fully accurate and complete pre-installation documents. I believe the death penalty is a consideration in some states if you receive the correct NEMA plug type schematic ahead of time. Of course, we installed exactly what the instructions stated, and of course, they were wrong. This isn't a knock on Bruker. It's the law. 

First impressions: Ease of use. do I write this without coming off as arrogant or stupid or both? This instrument isn't anywhere near as finished as hardware from other manufacturer's gear. Ease of use was not at the top of the design list. If this is your first mass spectrometer, I think it's going to be rough. 

If you are a biologist and the mass spec is just one of 15 tools you'll use during the week and you don't have a dedicated mass spectrometrist? 

Let's start with hardware first. Swapping from your ESI to your Nano source requires tools. You don't pop two hinges and swap them out. You'll need a tiny hex wrench (1.25mm, I think) there are multiple small parts that will be devastating to lose or break, including tiny gold o-rings and spring. It is very very easy to break your nano column while both installing and removing the nanospray source. It is reasonably easy to instally your nano emitter incorrectly, which you'll only find out that you did after you've fully installed your nano source, requiring you to take it all apart to reseat the seals on your source. You test that seal by blocking a filter on the completed source with a (GLOVED; there's EVIL STUFF IN THE FILTER) finger tip and monitoring the drop in vacuum pressure. 

Fortunately, you will only be swapping the sources every single day, so your chances of making mistakes will be rare. This can be minimized by putting evil fluorinated compounds into a filter so you have a spread of lock mass compounds into your nanospray at all times. I'm putting up a little sign to remind everyone. Gloves if touching the nanoESI. Huge shoutout to Gabriela and Brett at the UC Davis core for providing this secret protocol to me before I broke every NanoLC column in the entire mid-Atlantic taking the source off every day. 

Edit 7/21/21: You don't need to use the evil compounds for your lockmasses. Bruker has suggestions for lockmass compounds for proteomics that won't kill everyone. 

First impressions: Vendor software

Impressively, the TIMSTOF is compatible in an almost plug-n-play format with every vendor HPLC. If you can find the drivers for the instrument they load up well. Without question, the EasyNLC works better on the TIMSTOF than on any Thermo instrument. They communicate digitally and you have better control over parameters. Weird. 

Interstingly, the hardest thing to find in any of the vendor software packages is a mass spectrum. Chromatograms and mobilograms make the front page of most of the software that must be open while you're running the instrument. Finding a mass spectrum requires around 10 button clicks. I think someone forgot to tell the developers what this thing is. You can, however, eventually find one, but the software "Data Analysis" will be very annoyed that you figured it out and will ask you if you want to save changes when you close the software. 

Edit 7/21/21: The <Ctrl>+ Click button is your friend. Navigating around in Data Analysis isn't the most intuitive thing in the world. Fortunately, Mann lab seems to have notice this and fixed it with this fast and intuitive free software! AlphaTims to the rescue! 

First impressions: Performance

THIS THING IS FAST. Ricky Bobby drafting off a slingshot fast. Sloth escaping after burning down a hospital fast. FAST.  120 MS/MS scans per second at 40,000 resolution FAST. 

And the sensitivity is there. You wouldn't think that it would be, but it is. TOFS aren't sensitive. That's most of the problem with them, right? Holy cow. When you extract 500,000 MS/MS spectra out of a 60 min run with around 50ng of peptides on a column, it's easy to think "why was I cursing so loud about how hard it was to get this thing to show me a single MS/MS spectrum?" 

There are some really cool features hidden in the instrument methods. Best I can tell, mostly undocumented. There is a cool preprint from those Max Plank people where the authors state "functions of the instrument are largely unexplored'. I think I have a 1/4 finished post about that somewhere. 

First impressions: Compatibility with tools

This is getting better all the time, but if you find the fact that there are over 1,000 proteomics software tools in the world a little daunting, this might be an instrument for you! Very very few of the tools are compatible with the data from the device. Frustratingly, some of them will look like they're processing the data, hang out a couple days heating your office, and output gibberish. 

Things that don't work:
ProteoWizard (ouch. yeah. that one hurt. I figured I could just convert it to anything I wanted and reopen all my tools) 
Edit: 7/21/2021: ProteoWizard does work! You just need to update your versions. There is even a PasefMGF drop down for conversions. 
Morpheus - weirded out by this one. Even a Bruker generated mZmL crashes for me, but it might just be me. 
Actually, it could just be a poor mZmL formatting thing, so I won't list any others till I have a chance to put more time in. 

Things that do work: 
FragPipe/MS-Fragger (in some functions; no TMT, etc.,) 
MaxQuant (also appears to work, but not for TMT)
Skyline (very recent addition/update) it ever slow, though! 
Several commercial software packages; Bolt/Pinnacle, PEAKS, SpectroNaut, Byonic, what is Robin Park's software called again? IP2? It might be called something else now, and it can work in real time!  I meant to check that out, but busy busy busy.  

Again, this is just a first week with the instrument and I'm sure my impressions will change and evolve. There are a lot of pluses here. This monster of an instrument is enormously capable. It's fast, sensitive, and there is loads of opportunity to build new and exciting experiments, but it doesn't feel like a fully finished product. I think that early adopters are going to largely feel like beta testers. And if that's what you're down for, hell yeah! Me too!  

But if spending lots of time doing method development and maybe fabricating useful parts or testing less dangerous chemicals for calibrating your instrument, or writing patches to make your favorite tools work with your new million dollar instrument isn't what you consider a good use of the minutes you have left on this planet, this might not be what you're looking for yet. It'll be interesting to see what direction this new tech goes in, though! 


  1. What's the interscan and intrascan dynamic range like (especially compared to say the latest Orbis, eg Exploris?) My understanding (from a Waters tech support person though) is that one consequence of ion mobility is that it reduces the dynamic range....

  2. This comment has been removed by a blog administrator.