Tuesday, May 29, 2018

Let's check out the new PASEF TOF thing!

Have y'all seen this thing yet? I just got to see a great talk on it and I'm ready to say that -- for bottom up proteomics -- this is the most powerful Time Of Flight instrument we've ever seen.

EDIT 6/9/2018: I've spent some time talking to smart people about this thing and I've -- overall -- felt kinda dumb about this post for a number of reasons
1) I got really annoyed about my inability to see the instrument data or process it and kind of went ranting
2) The rants on this page aren't based on real facts, just some grainy marketing info on this device. I should really withhold judgment on this thing until I can see some real data -- however -- I definitely don't want to seem like I'm endorsing it until I do. I've seen MadMen. Marketing people are scary.
3) When in doubt we should be encouraging competition in the marketplace. Just look at what the resurgence of AMD has done in the PC marketplace. Intel has real competition for the first time in years and we're back to exceeding Moore's law for microprocessor performance! Maybe the same thing can happen on the mass spectrometry front.
4) Now I'm afraid people think that I've actually ranked my dogs in the order in which I would eat them if I had to....

I'm going to leave this post in place as a reminder to myself to do think before I post. The walkthrough on how to look at Bruker .d data in MaxQuant does appear correct, if you use the new version. And I'll put up a new post later when I actually review some data.

There are a lot of bells and whistles in the instrument, but probably the central technology is PASEF which was described here in 2015. TOFs are FAST. Screaming fast. But -- sensitivity is a serious problem. You need to accumulate some ions before you shoot them down a tube toward a detector. -- especially one that is several feet away to get anywhere near the sensitivity of a quad, a trap, or an Orbitrap.  PASEF and the TIMSTOF allows accumulation of ions before shooting them and that can bring up the sensitivity a lot. It also allows parallel filling and that speeds everything way up.

Okay -- so -- let's not go into details, but someone sent me a Zip file with some data from one of these things. I'm normally not nervous about science stuff -- but, holy cow, it is really hard to find data from one of these things. I'm sure that with brand new technology it probably takes a while to get everything formatted right for public uploads and things, but when I do see some uploaded via ProteomeXchange partner, I'll feel less like this....

If you get into the file -- you'll find it is an SQLite format (interesting!). That is what the processed data from Proteome Discoverer is -- so if you want to open it you will need to go and get a free copy of SQLite -- you'll need 4.5 or newer -- and if you don't want to hunt for it, you can get it here for Windows 64-bit.

You'll definitely need to check first to make sure you have a .NET framework that is this new. Here is a guide for how to do that. This will require administrator access. When you get there you'll see something like this, probably. If you do, you'll need a .NET framework upgrade.

To get that, Google "Download .NET framework 4.5" and verify that you're on the legit https:/ microsoft.com site (lots of fakes out there). Close everything, upgrade, reboot and come back to this. If you're on Windows 10, this should already be preinstalled. No problem.

To process it you'll need a recent version of MaxQuant, the newest version of PEAKS (haven't checked this one) or -- it sounds like the most recent Mascot! Easy! I'll go with MaxQuant. We need to get reintroduced anyhow.


Okay -- let's assume operator error, put a note on the MaxQuant discussion board and try something else!  EDIT: See bottom of post for correct MaxQuant version to open this data.

Here's a cool video that talks about the instrument! Let's check it out!

This is a really solid video. Dr. Rather explains how the PASEF thing works, and how we have to think about TIMS data in a different way than we're used to seeing. Being a guy who had the first MS/MS spectrum he ever published tattooed on himself, there's obviously some things I'm interested in seeing. At 22min and 37 seconds you'll find a spectrum to check out.

I drew two question marks here. Let's look at the one on the left first!

If you go to Google and type in "proteomics red elephant" it will take you to a blog post I wrote about a tool I use every single day, the pHpMS webserver. I love this tool.

If I punch in the sequence for this peptide in the Fragment Predictor it tells me this instrument is getting the right doubly charged mass

888.94 compared to 888.9329 -- I'm not going to be a jerk here, for real. Yeah -- I like 4 decimal places. Honestly, in this range we're only accurate (without post-acquisition recalibration) to the 3rd decimal, even on a Fusion 1. Even if we assume the very worst, that the measured mass was 888.9449, this is only 13.49 ppm off, right? I'd rather eat my dog that I like than send a peptide to a collaborator where my MS1 was off by 13.49 ppm, but this is the very very maximum, it could easily be 888.9351 rounded up, which would only be 2ppm out. For reference sake, I wouldn't eat my other dog for less than 8ppm.

How do the MS/MS fragments line up? It's tough to tell with it being all no numbers and stuff, but we can extrapolate. y10 should truly be 1203.5423 and y14 with z=2 (**in the figure) should be 853.4143. If we assume that gap between the scale markers at 1200 and 850 are the same --

---Its probably just the visualization tool and a low res image of a low res image, right? y14 sits precisely on the 850 marker and y10 does not. The visualization tool used here could be at fault for sure...as bad as my eyes are I could be mistaking what appears to be a 3 proton gap and a nonexistent 3 proton gap. I don't even know why I'm still talking about this. I'm sure it's fine.

Let's go somewhere else. What about phosphoproteomics!?! Wait. I didn't do the other question mark. Ummm...let's come back to why we fragmented the same unmodified peptide 3 times...later...or not ever....
(Edit: 6/9/18) Surely it can do monoisotopic averagine modeling, right? Q-TOFs 10 (15?) years ago could do that.... Ummm...wow...got some vague answers when I asked. I'm a little hung up on this one. Monoisotopic precursor selection (called MIPS or PeptideMatch by some manufacturers) is a big deal. Chances are you don't know your instrument is even doing it. If you want to see whether it's just a dumb button or not, go in, turn it off and run that sample again.) Actually -- new topic. Go to this paper from Dave Muddiman.  Turning off MIPS cut the IDs in half, and that version of MIPS was not very good -- at least compared to today's algorithms. (That one would throw out SILAC 'cause it couldn't resolve the isotopic differences. Buzz me if you need a reference on that, my memory is problematic, but I'm seeing some hazy details -- something about a smart ultra-marathoner from somewhere in Texas who showed that conclusively and the manufacturer fixed it...MIPS is that important! Dr. Bob Swaim would know exactly who I'm talking about, so I'll email him if anyone asks me about it. If you know him, email him for faster turn-around)

There is this REALLY cool thing and it's called the PhosphoProteomics eBook for this device. You can look it up on the Google. There are two spectra in it --- of basically coeluting isobaric phosphopeptide species (there was an AMAZING talk at ABRF about this from [correction -- Brian Searle gave this talk -- consulted my notes] about how often biologically relevant isobaric phosphopeptides elute together, but I haven't verified he's published what I want to ramble about yet). The isobaric species in the eBook look like this --

Here's the pitch -- without the ion mobility, you'd never be able to tell these two isobaric peptides apart. They'd coelute, muddy the water and you'd never figure them out.

Back to my outdated obsession with accuracy in scientific instruments in the year 2018 (bear with me, please) we have two isomers and one has a mass of 546.2626 and the other isomer has a mass of 546.2536

PPM calculator says --- 16.47ppm....I really like my dog. For real....who wouldn't!?!? Check out this dumb Mufasa thing he does sometimes. That is a really dangerous place to stand.

...but if you think that I wouldn't choose to eat that big majestic monster before I'd tell someone I had two isomers at >15ppm apart and the only evidence I had to back it their identity up in a human digest was fragmentation data scaled to the nearest 500 Da and one of the isomers has no evidence of neutral losses whatsoever.... I'm joking, of course. I'd probably eat Bernie if it came down to it, but definitely not Gustopheles.

Edit 5/29/18: Crazy idea I need to leave here so I don't forget. Could we simulate the 2 phosphopeptide fragmentation patterns and then see if Ascore or phosphoRS could tell them apart?

Wow. That was SO MANY WORDs. Not sure why I wrote them all now. Let's sum it up.

At ASMS we're going to hear a lot about a really cool new parallelized ion mobility trapping TOF thing.

It's probably the best TOF for proteomics the world has ever seen.

Yeah --- it can get upwards of 100 scans/second -- but at that speed it can't do sophisticated calculations like monoisotopic precursor selection on the fly. It also may not be capable of dynamic exclusion the way we're used to seeing it. And -- the mass accuracy isn't what we're used to seeing these days from fourier transformation or even modern TOF based instruments.

I'm not saying it's a bad tool. We've discussed how it might fit into our workflows at my day job, but there is this inherent danger in the explosion in value in the mass spectrometry commercial space when business marketing teams get to run ahead of peer reviewed science. There's a lot of flash around this big new box, but -- man -- there sure isn't much on the evidence side yet...

Update 5/28/18: MaxQuant was released on 5/27/18. It has native PASEF TOF support!


  1. Hi Ben,

    you are able to adjust the TIMS scan time which determines the number TOF scans within a TIMS duty cycle. Results have shown that 50ms are much more benefical for number of unique precursors compared to higher TIMS duty cycle time. You might want to check this out https://www.biorxiv.org/content/early/2018/06/01/336743

    Regarding the monoisotopic isolation - Since you have the ion mobility dimension they is a fewer need to isolate monoisotopically that is the major advantage of the technology.


  2. Ben,

    Did you ever get a chance to run the PASEF data?