Targeted quan on the QE part 3

Welcome to my monologue on the many methods for targeted quan on the Q Exactive.  Yes, I honestly took the time to make a Super Saiyan Q Exactive.  No, I don't know what is wrong with me.  Yes, I'm starting to worry.

Check out part 1 here

And part 2 here.

Now, while my sanity was still intact and before I let slip that I watch early 80s Japanese cartoon shows, I said that in part 3 we'd start looking at Sensitivity and Specificity.  This is the big stuff.  As in the previous methods, we're always trading something for others.  Ultimate sensitivity takes time.  Ultimate specificity takes resolution and/or fragmentation, which also takes time.

Method 1:  T-SIM-ddMS2 (also called parallel reaction monitoring, or PRM)

The crazy amount of sensitivity (btw, I think the official Thermo specs for the original Q Exactive is that a T-SIM is 13x more sensitive than a full scan.  This depends on the complexity of the sample at hand.  On complex stuff it is WAY more of a boost than that.  The official specs were derived from caffeine on cal-mix.  Sorry, I meant to mention this in post 1) PLUS as much specificity as you want based on any resolution you want + fragment ions.

Advantages:  Knowing without a doubt in the world that this is your peptide of interest.  Crazy sensitive at the MS1 level.  Very sensitive at the MS2 level.

Disadvantage:  Cycle time.  Even at the lowest resolution setting, we are looking at 128ms per target.  This can be offset, cause WE CAN MULTIPLEX IT!!!  WooooooHooooo!!!!  The MS/MS event is dependent on a trigger.  If we don't have the target of interest we don't have to add the second 64ms event.  But, on super low abundance targets we might not get our MS/MS event to trigger.

ENTER PRM #2

BOOM.  Get around the triggering requirement and make your QE act just like a triple quad running MRM mode, except with high res accurate mass (and, I would still argue, every bit as much sensitivity, if not more, than any QQQ when you use maximum fill times.)

Disadvantage:  Cycle time.  Cycle time.  Cycle time.  Cycle time.  This is the slowest method that we can run on the QE.  128ms per target is mandatory minimum.  But if you are looking for a phosphorylation event on a 1 GTPase that is expressed at 50 copies per cell and you have to find it, this is your method.  Ultimate sensitivity, ultimate specificity.  Now, if we want to get into the super low abundance ions here and use maximum fill times, we are talking as much as 6 seconds per target!  Is this really practical for all experiments?  Not really, this is 5-7 scans points across a 30-45 second peak.  It is a nuclear option for when we need triple quad or better sensitivity to detect a target at the lowest possible levels. 

Okay, I need to get to the gym and get back to my real job.  Part 4 will be on Data Independent Acquisition methods.