Targeting on the Q Exactive: Which method to use and when?

I don't even care to keep my love of this instrument under wraps.  I'm more of a discovery proteomics oriented guy, but this stuff is so easy on the Q Exactive that there isn't all that much to talk about.

More and more I see the Q Exactive replacing more traditional (QQQ) platforms for validation and targeted measurements.  Don't get me wrong, a triple quadrupole instrument (QQQ) is a great thing to have, but you can really achieve a lot of what it does by subverting your Q Exactive from discovery for a little bit.

You have these options on the QE for targeted quantification

1.)MS1 based quan - full scan
2.) MS1 based quan with targeted ddMS2
3.) AIF (all ion fragmentation)
4.) MS1 - AIF
5.) Targeted SIM (single ion monitoring)
6.) Targeted SIM - ddMS2 (triggers MS2 if something in the SIM matches your target)
7.) Targeted SIM - Targeted MS2 (performs SIM and then targets everything in the SIM window for MS2)
8.) Targeted MS2 (called PRM, for Parallel Reaction Monitoring)
9. DIA (data independent acquisition)
10.) msXDIA (multiplexed DIA)
11.) pSMART (MS1 followed by an intelligent number of DIA windows)

Eleven methods!  And this is without really considering multiplexing!

I'm going to divide them up into 2 groups:
1) Maximum number of targets
2) Maximum sensitivity

Maximum number of targets, lowest sensitivity:
Methods 1-4 and 9-11
On a normal chromatography gradient (60-120 minutes is what I consider "normal") if you want to look at, say, more than 200 targets, you need to go with one of these.
In order of sensitivity, from most sensitive to least, this is the order I'd generally go with.

11 & 2 > 1&3&4 > 10 > 9

Where I'd consider MS1 based quan with targeted ddMS2 to be equal to pSMART in terms of sensitivity and regular old DIA is the least sensitive.  Honestly, they all come pretty close and a well tweaked DIA may beat out a not-as-well optimized pSMART.

Lowest number of targets, highest sensitivity:
Methods 5-8.  Super sensitive but you can only look at a few targets at a time.  For reliable quan, I use the cutoffs Mark Chance's lab uses at Case Western for their clinical proteomics samples.  I want to see 12 measurements across the peak to trust that I have a good quantifiable peptide.  For this equation, the realistic fact is this.  More sensitivity = fewer targets.  In order of sensitivity

7 > 5 &6 & 8.  In my hands (old data...) 7 is more sensitive because you have 2 chances to get your ion of interest for measurement.  You get the chance to measure it with just SIM and you get the chance to measure it with PRM.  Half the number of targets.  Otherwise the rest are the same.

Consider this really simple example.  You have 30 second wide peaks.  At 17.5k, the QE can get about 13 scans per second.  If we use the cutoff above, 12 measurements sample (heck, round to 13 to make it easy, this means -- at most-- we can really look at 30 targets at a time.  Now, this is also assuming that the limit here is the speed of the Orbi, for this to be true, the fill time needs to be 50 ms for the QE or 57 ms for the QE Plus or QE HF.  A lot of times, you aren't targeting something that you can pick up well at 50 ms fill time.  And some people might argue that 17.5k resolution MS1 scan isn't enough resolution to clearly say that is your target of interest.  If we go to a more realistic example of 35,000 resolution, and a fill time of 114 ms for QE then we now have to cut the maximum number of targets we can quantify ...at once... in half, to 15.  At 70k, we'll have to drop to 8.

Tricky, right?  For a really good analysis of this including illustrations that explain this a whole lot better, I recommend you go to Planet Orbitrap and download the iORBI 2015 called Maximum Peptide IDs by the great Tara Schroeder.  I can't direct link to it because you have to be registered and logged into Planet Orbitrap to follow the link.