Thursday, March 31, 2016

Case study -- Beautiful looking data but NO peptide IDs!!!!

...Sherlock Pug...

Anyway!  I recently got involved in some sleuthing helping a friend solve a really weird mystery and he gave me permission to share this in case any of you guys run into it.

Background: GREAT lab. Q Exactive Classic that is approaching historic levels in terms of the number of papers this one little box has turned out. Absolute work horse that has pretty much been calibrated and PM'ed and otherwise hasn't stopped running in 4 years.

Big study underway. Fractionated samples through a time course or something. 400GB of RAW data or so. Ran through a weekend and a holiday break. Why not. This thing has never had a problem and we know from lots of historical evidence that the calibration will be steady throughout the run.  And this instrument is always lockmassed on our good friend 445.120025 (QE rounds to "3", of course)

Go to process the data --- no peptides ID'ed! None. Check the TIC? Fantastic!  Check the MS/MS spectra -- tons of beautiful spectra. WTF!! (what the phenylalanine??)

There are a lot of steps in a proteomics pipeline where things can go bad, right? But the RAW data looks fine, so maybe Proteome Discoverer isn't communicating with the Mascot server correctly? Maybe the Mascot server is just mixed up? Maybe someone switched the FASTA database (don't laugh, I've seen that one, LOL!)

I'm lazy and don't have a lot of free time, so I start with one file. I run it through Sequest on my server. Same results.  Time for Preview (which I run through PD, of course!)



HOLY COW!!!  Wait, what? We're 65ppm off at the MS1 and 64ppm off at the MS/MS??  Is this the best file I ever ran on my QTrap? (LOL. Hey, I loved that QTrap and built my entire career on it, but you don't buy a QTrap for mass accuracy. You buy it for...well, we bought ours cause it was free! True story!.)  No, this is Q Exactive data and 65ppm is nuts

More investigation. This lab had power outages do to a recent severe storm. The Orbitraps are all on UPS, so they never went down. But the climate control? Well, it lost its mind.  Turns out the room where the Orbitraps are may have gotten heated to over 90F (that is over 32C for you people who use intelligent units of measurements. Slackers. Anyone can count by 10s!  In 'Murica we like to challenge ourselves with arbitrary historic units!)  Over 90F...maybe more than once.

So...if you've got an LTQ Orbitrap in a room and your room goes from sauna to freezing a couple times, chances are nothing is going to happen. There is a huge water cooling system on that bugger and its gonna stay at/around 26C. A Q Exactive, however, has no cooler. It compensates calibration by the temperature and there are limits.

Also, lock mass on a Q Exactive can only correct mass errors within 20ppm.

Okay, mystery solved!  How do we fix it?

Well, Preview/Byonic's recalibrator has no problem with it. Lets try another fast recalibrator!!!


Thank you Mechtler lab!!  Now, normally this thing works without a hitch. Boom!  Recalibrated MGF output. In this case, however, we uncovered a limit to this awesome free resource (this is at pd-nodes.org, btw).  65ppm is too far out, it appears.  I fixed some stuff that was around 20ppm, but that has been my upper limit so far.

What else? Now, there are plenty of tools out there, but I don't know all of them.

One tool that no one seems to know about is a free tool from Thermo (I didn't know about it, shoutout to Detlef for introducing me to it). It might be already installed on your instrument.  Some engineers put it on at install, especially on the LTQ Orbitrap instruments. If you don't have it you can get it at:  https://thermo.flexnetoperations.com

The tools are called FTPrograms. There are several cool things in there, but one of them is called RecalOffline. If you install FTPrograms, Windows may not recognize it as a new tool and highlight it. You'll have to use the search bar to find RecalOffline, but then you can make a desktop shortcut to it:



Okay, here I grabbed a random file and tried to anonymize it. This is just an example and I've got a meeting to get to!

So you give RecalOffline a mass to choose. Here I put in my polysiloxane 445 and then you give it a mass tolerance. I cranked this up to 70ppm just to see if it can take it. And you have other setting controls. It will then go through every single MS1 scan and adjust the calibration so that you've essentially lockmassed the file.

Worth noting...um...this can be very slow. Do this on an old Dell you've got sitting in a corner that you aren't sure why you still have. Or on something real fast (SSD, for the win!). You can also use the Slicer function to cut out a smaller section of the file and then Recal it to reduce your processing. And as far as I know this GUI can only be used for one file at a time. I believe there is/was a way to automate it from the CMD prompt, but I don't know if that feature is still enabled or how to do it or if I just made that up.

And please note, I'm not questioning anybody's skills here. These guys know what they're doing. If you walk away on a Friday and there is no alarm to tell you that your buffers on your LC are boiling, this can totally happen to you. I shared this anecdote with some of the members of the great Thermo COE team and 2 of them had seen something similar happen in their years on the road.

What I want to show you is that all is not lost. Chances are you can totally save those files, and these are tools that can do it.  BEN, SHUT UP AND GO TO YOUR MEETING!!!


2 comments:

  1. Hi Ben,

    We used the RecalOffline tool in our recent paper on RePLiCal (a retention time standard, http://pubs.acs.org/doi/abs/10.1021/acs.jproteome.5b00988), using the standard peptides as reference to lock mass to, and it basically reconstructed the raw data (the siloxane ion at m/z 445.120025 isn't always present as discussed in this paper -> http://pubs.acs.org/doi/abs/10.1021/pr100780b, so it can be useful to have reference peptides throughout the gradient to work with). RecalOffline really is a useful tool to save experiments, which could be costly in terms of time. money and/or sample if the analysis failed!

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  2. Hi Ben, I am desperately looking for a way to download FT programs for RecalOffline but no luck.Can you help here?

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