Hi Dan,
Skyline only considers the monoisotopic fragment ion peaks, and you are right that the only reasonable explanation for you your signal goes up when you widen your isolation window is that you begin including the M+1 precursor, which will still produce monoisotopic fragment ions when the single 13C is not included in the fragment.

In the early days of Skyline, Dan Martin once (8 years ago) proposed to Mike MacCoss and me that this effect was visible in SRM on a triple quadrupole instrument and that Skyline could help make it a mainstream practice. I don't remember his lab ever publishing that finding, but of course a triple quadrupole doesn't really have any way of deisotoping. There also, opening up Q1 simply allows the inclusion of M+1, which contributes signal the monoisotopic fragments. I know Mike didn't see this as worth our pursuing.

As you note, it will also increase the probability of interference, and potentially make other computation more complicated.

Triple quadrupole instruments have been in use for a long time. Given that the effect is just as achievable on them and the fact that it is not widely used in SRM may be a good indication of how useful it is in the end.

Maybe I can get Mike to contribute his thoughts on this later. Thanks for posting to the Skyline support board.

--Brendan

Hi Dan,
I will add my $0.02. You are correct that decreasing the resolution of the quad isolation (i.e. increasing the isolation width) will increase the intensity of your targets. This is definitely an old and common trick to improve sensitivity in the absence of interference. It is also partially why DIA has been enabled. While DIA is much less selective, the transmission of a 5 m/z isolation window is much better than a 1.4 m/z isolation window. Think of it as a pipe, the larger the pipe the faster you can move water through it. The same thing is true with quad isolation. You see the increase in signal because of the reduced ion trap fill time as the signal is a function of the current/fill time. So you don't have any more actual signal you just got there in less time.

You're also correct that if you isolate the entire isotope distribution that you will get isotope distributions on the product ions that could give you more ion counts if used correctly. We have been thinking about ways to use the entire isotope distribution like you said to deisotope the spectra and then extract just the signal that is reflective of the entire isotope distribution. Nick Shulman on the Skyline team did this previously in a different tool called Topograph (http://www.mcponline.org/content/11/11/1468.long).

The problem comes when you are isolating only part of the isotope distribution. Then the isotope distribution on the product ions is not straight forward to predict. Because of this our first effort to do this will almost certainly be on the MS1 signal. Nick has been thinking of ways to combine the deisotoping with a fit in the time dimension too so that we can be better at handling incompletely resolved extracted ion chromatograms.

Brendan is right that I am generally not a big fan of decreasing selectivity as part of the routine targeted analysis. My preference has often been to just use product ions that don't have interference as opposed to adding a much more complicated signal processing step that would add variance (noise) to the measurement. Increasing the ion counts often increases the precision as long as it doesn't come at the expense of chemical interference. Additionally on trapping instruments the AGC reduces the fill time and it can let you scan faster without sacrificing sensitivity. That said, we are becoming big adopters of DIA workflows and the line between DIA and PRM is becoming blurred with methods like MSX (http://www.nature.com/nmeth/journal/v10/n8/full/nmeth.2528.html?foxtrotcallback=true). So we are considering all of these classic signal processing methods.

Thanks for the post. We don't have a solution that can be used for deisotoping within Skyline at the moment. That said, it is something that is very much on our mind.

-Mike

Hi Mike, Hi Brendan,

thank you very much for sharing your thoughts on this, I really appreciate the insights.
 I will play around with the isolation width and the isolation width offset to have an narrow width but still include the first isotope when collecting ions.

Best
Daniel