Wednesday, September 17, 2014

Targeted quan on an LTQ Orbitrap part 1: SRM/MRM



I get this question all the time: "Can I do targeted quan on my LTQ Orbitrap?"

Answer?  Of course you can.  If you know what you're doing there isn't much you can't do with an LTQ Orbitrap.  There is a major limitation to the targeted stuff, though.

You can have maximum sensitivity and selectivity OR you can look at a ton of targets at once.  You can't do both.  Tara Schroeder said "you can have breadth OR depth," which would be catchy, but I have trouble saying it out loud.  Seriously!  Try saying it 3 times fast.

If I want sensitivity on the LTQ Orbitraps the LTQ is the way to go.  It is simply more sensitive.  The ions are coming in the front and get there first.  It also uses detectors where the ions make physical contact.  The LTQ is also faster than the Orbitrap.  This is less of an issue with the speedy Orbitrap on an Elite, but is very very obvious on a Discovery, XL or Velos instrument.

We can also get a different kind of selectivity.  We can do SRM/MRM and then do our quan on the fragment ions!

First select a generic MS experiment (don't do data dependent):  Also, click to expand pics.


For scan event one you get two choices.
Full scan for targets that vary a lot by mass.
SIM scan for more similar targets and sensitivity.  You can also have multiple SIM ranges.

For the MS/MS you have a couple of options.  If you check that little box at the bottom "Use MS/MS Mass List, this is not a data dependent experiment.  This becomes akin to fragmentation on a QQQ instrument.  We're gonna fragment everything within the ranges specified.  Going to the Mass Lists tab at the top of the screen will give you a new option under this tab and a new function for the table.


Its called (+) MS/MS l.  Why?  Who knows.

The important part is that you get to put targets into this table at the bottom AND you can time them.  Here I'm targeting two peptides with 2 different collision energies.  Since I have 1 event set up and I only have 1 event timed at a time, this is perfect.  The instrument will do my MS1 scan followed by a fragmentation of anything in the range of my parent mass, then go back to MS1.  If we need to look for more than 1 target at a time, we'll get MS1 followed by MS2 of target 1 then MS2 of target 2.

Small target lists are best here.  We shouldn't look at more than 10 targets at a time under most circumstances.  In the tune file you can start by matching your fill times to your scan times (say 100ms for earlier instruments, more like 80 for the LTQ Velos systems.  If you need more sensitivity, crank up that fill time.  200ms gives you twice as good a chance of getting good MS/MS fragments than 100ms.  The best part is that you don't use the extra fill time unless you need it.  For target values you'd be fine anywhere from 5e3 through 1e5, but I'd probably start at 5e4.

Does that cover it?  Again, there are more ways.  We have a huge amount of flexibility with an LTQ Orbitrap, but this ought to get you started on one way of using it.

No comments:

Post a Comment