Thursday, May 23, 2013

Recalibrate Thermo RAW files automatically

This is AWESOME.
There are several reasons that we might want to go back and recalibrate our RAW files.  Sometimes you find out after a big experiment that someone forgot to calibrate the Orbitrap (not me, but other people!  I'm a super calibrator.  Sometimes you find out you left lockmass off.  Or you have an Orbitrap XL or Discovery and lockmass adds to your cycle time so you leave it off to dig a little deeper into your data.

Until now, there was only one way to recalibrate your RAW files and it required that you use the command prompt, you had to do it one at a time (single core, even) and it doesn't work with anything newer than a Velos.

Then we get this new free node from the Mechtler lab.  You download it, follow the simple installation instructions and then set up the workflow as shown above (there is another formation on, but I couldn't get it to work myself), and you recalibrate your RAW files.

I pulled an old Orbitrap XL file from my postdoc and gave it a try.  I never used lockmass due to the cycle time issue on this instrument, so it made a perfect test.

206 peptides in the fraction (ran with no modifications):

Run 1:  Average mass variation:  4.65 +/- 1.15 (not too shabby for 30,000 resolution)
Run 2: (Post calibration!):  0.87 +/- 1.06

BOOM!  I exported it as an MGF file and then reimported that one back into the exact same workflow.  Sub-PPM mass accuracy on an XL without lockmass?

I don't think I have to spend a lot of time on this.  I hope the immense value of this amazing tool is apparent.  How many good peptides get dropped because they are 11 ppm out?  I'm curious, because I think there are a lot.

Another idea that just popped into my head:  If our goal is to get into the sub-ppm mass accuracy range and we can get it at 30,000 resolution or even 60,000 resolution, would we be able to save cycle time on experiments we would have ran at 100,000+ by just running them at lower resolution and then recalibrating them?

So much potential here.  I can't wait to experiment further!  Thank you Dr. Mechtler and lab!


  1. Hi Ben,

    Is there a publication that describes this utility and a download link? It looks very handy!

  2. Unfortunately, there hasn't yet been a publication out of this group. There is information and the downloads are available at

  3. Hi Ben,

    I understand I am a bit late to comment but I'm interested in the other way you mentioned that could be used to re-calibrate data from older instruments including the Velos. Is the program still around and does it have a name?


    1. Chris,
      Sure! It is called "ReCalOffline". Chances are it is actually installed on your instrument PC. If not you can go to any Xcalibur install file and find FTTools and install that whole thing. It'll put RecalOFFline and some other neat little executables on your desktop. Let me know if you can't find them -- or don't have access to the Thermo Flexera FlexNet where you can download them and I'll send you a download link for a version that is compatible with (almost) any Orbitrap files.