Saturday, June 23, 2018

Worse than boring post -- Trying to find specific MS/MS scans in Xcalibur!

Imagine this strange scenario -- you have a peptide that you discovered with your search engine and you just want to take a quick look at it in Xcalibur.

You have the assigned monoisotopic mass from your search engine -- all the way out 4 decimals.

You are easily able to find it by XIC with ranges like this --

And now you want to take a look at the MS/MS spectra in Xcalibur -- you open up ranges for the MS/MS spectra triggered -- and it isn't there -- there is some stuff that is close -- but nothing exact...

Ummm....where'd the MS2 go? 

Turns out Xcalibur has it listed twice. 

If you are just scrolling along through with no range filters applied you'll find the top spectrum (T), but if you go along with ranges you'll need to hunt down the bottom spectrum (F).

Note that this is the exact same MS/MS scan -- but the top one is the mass you are looking for and the bottom is the one you'll have to use in ranges to find it. How fun is that?!?!

I have no proof of this -- but I believe that the bottom mass (and one that is assigned in ranges) is the preview scan mass. The Orbitrap doesn't complete it's full MS1 scan before it starts acquiring ions that it is going to do MS/MS on. We don't want to waste that much time.  Partway through the scan it's already created a list of ions that either passed MIPS (also called PeptideMatch) or the minimum intensity and charge cutoffs you provided for it and then starts working on getting them.

In this case -- this wasn't hard to find. We're off by just a tiny amount. And this is the Tribrid. This issue is much worse on the Q Exactives.  Here is a typical example.

A standard tactic I employ is opening an MS/MS range for every ion mass close to my ion of interest.

In this case I can find the scan on the second attempt

The difference here is about 0.02. Off the top of my head, that sounds like about 30ppm. Neat, right!!??!  My second favorite part about it is the fact that the ranges aren't listed in --- numerical order. 

This post is worse than boring. I honestly thought when I started this that I'd come up with a clever solution for finding what I was looking for -- and I still don't have one...

It is worth noting that Xcalibur is kinda old. And FreeStyle is probably meant to replace it in the near future for good reason.  Maybe FreeStyle is the solution?!?! 

EDIT: 6/26/18. Thanks for the tips in the comments, y'all!!  I will try tracking by scan number. Unfortunately -- and I should talk about this later -- sometimes I'm trying to track Minora features that don't provide you with scan numbers. 

I did try FreeStyle and it does the same thing -- 

--- but it does it SO MUCH FASTER that it might become my go to almost immediately with one really weird outlier -- data from our Elite only gets 2 decimal places by default while the Fusion gets 5....

...which is probably my fault, but I can't seem to change it...FreeStyle automatically recognizes the correct settings for the Exactives and Tribrid and that saves me a lot of steps. 


  1. Maybe it helps to look up the desired MS2 spectrum just by using the scan number in this case. Usually, search engines will provide you with this, too

  2. I always look at the scan number associated with the ID from the search, then display the chromatogram in stick mode, with scan numbers displayed. Makes it very easy to walk through and select the same scan in QualBrowser.