Should you be using lower collision energy for PRM experiments?

Okay...so I was running my mouth again about how PRMs on a quadrupole Kingdom trap system could beat SRMs for a QQQ and had to blow a weekend in lab running stuff to prove it to a bunch of skeptical people.

Caveats here for why I made this very costly dare (I probably only have a few thousand weekends left in my whole life after all...)

This researcher has only one peptide that he can use to confirm a positive outcome for this assay. One peptide. (Plus controls and whatever, of course).

There will be pressure for the LC-MS assay to be as short as possible.

The matrix is...whole...digested...human...plasma (or serum or whatever. A friend told me there was a difference yesterday and I still don't know what it is)

If you've got a protein you can get 3 peptides from for this, okay -- a QQQ might be the better choice for this assay -- but if you've just got one? I'm going PRM all day and never consider the QQQ.

I can't show the actual data cause I signed a thing that looked seriously scary. But I can tell you this -- there were so many peptides in the close mass range of this peptide in the digest on a 20 minute gradient that there was no way I could even trust SIM -- even at 70,000 resolution (max I had on the instrument I used) -- nope.  HAD to be PRM.

And -- when I was looking for fragment ions for my quantification (btw, I just extracted with Xcalibur and I believe it sums the fragment intensities rather than averages them -- but I'm not 100% - the peptides look great in Skyline as well) there was enough coisolation interference at with a 2.2 Da window that I couldn't use anything in the low mass range at all.

With this information I created the super-scientific scale that you see at the top of this post.  I really had to go to high mass fragment ions for specificity in my quan (and the best possible signal to noise!)  How complex is the matrix -- that with a 2.2Da isolation window there are smaller peptides you can't trust -- extracted at 5ppm...?

And, you know what? I could boost the intensity of these big fragment ions by dialing the collision energy back some.  Not a huge boost, but dropping the nCE down to 25 might have picked me up 10-20% in this particular assay for this particular peptide. (Your results may differ)

Let's check some experts!

I went to ProteomeXchange and searched "PRM" and downloaded some RAW data at random from a couple studies out there....and...I totally "discovered" settings I should have been using the whole time....yeah...you should probably use a little less collision energy for your PRMs!

The first 2 studies I pulled...used...25! (PXD003781 and PXD001731). 2 other studies -- RAW files completed just as I was wrapping this up appear to have used 27.  We're at 50/50, but my peptide really liked lower energy.