Full Scan and PRM for quantification

[francesco2995]

Hi everyone,



I would like some clarifications regarding the PRM technique. I have always used this technique for the Orbirtrap Q Exactive Focus for quantification analyzes on food matrices. By doing some tests on a matrix, I realize that the full scan spectrum for a given molecule has a decidedly better quality, as well as being more intense (but I think this is normal), for quantification compared to the PRM spectrum.



What I can't quite understand is why in PRM, a more specific and selective method, the peak is of low quality. I used a method already tested for other analyses: Resolution 35,000 ; N(EC) 20.40, 70 eV; Insulation width: 1.5m/z; Target AGC 1e5.



What could it be related to?

Thanks,

Francesco

[Gaetan Glauser]

Hi,

I can think of a couple of things which may have an impact on what you observed. Not saying they do, but at least worth considering:

First, in PRM you fragment the molecules; in other words, you spread the signal over multiple fragment ions. Compared to the precursor ion, the absolute intensity will always be lower for the fragments. It only becomes advantageous if the noise becomes even lower and/or if selectivity issues occur in full scan which can be overcome in PRM. In SRM/MRM with a triple quad, the gain in selectivity over SIM is usually important, but with high resolution instruments the mass extraction window can be so thin that the advantage of having precursor selection (with a 1-4 Da window in the first quadrupole) is not always beneficial.

Second, PRM is like a multiple product scan. If you set too many PRM events within an analysis, it is possible that the dwell time becomes too small or that the total cycle time becomes too long, both having negative effects on peak shapes.

Hope this helps

[lmh]

Did you use the same target AGC in both? I think the default AGC is generally lower in MS/MS events than MS

[lmh]

An addendum: on a QExactive-type instrument, sensitivity is limited by how many ions you can collect in the C-trap without space-charging issues. That's the target. SIM and PRM only make sense if your analyte coelutes with something else.

If your analyte runs on its own, a large proportion of the ions in the C-trap will be your analyte, even in a normal full scan in which all ions are permitted into the instrument.

If your analyte coelutes with a huge contaminant, and you are using a mode that lets all ions into the instrument through the quadrupole, then most of the ions in the C-trap will be contaminant, and sensitivity will be reduced. In this case, if you use a SIM or PRM method where the quadrupole is selecting only the correct mass, you can filter out the contaminant and fill the C-trap efficiently with the correct mass, which will bring the sensitivity back up to the level you'd hope for from a clean peak.

But PRM will then suffer a bit of loss because only some of the precursor ions will fragment to the mass whose chromatogram you extract.

I hope I'm interpreting the inner workings of the QExactive correctly. Someone please tell me if I'm wrong! Incidentally, when I write SIM, I mean "proper SIM" using the quadrupole, not SIM as in SIM-Scan which they use for their normal scans. In the QExactive, that sort of SIM is just a scan across a very short range, and there's the option (advanced user settings) to set up quite a lot of them in a single event, but they are still short-range scans taken from one trap-ful of ions.

[Gaetan Glauser]

Agree with lmh. If the AGC values are set with an order of magnitude difference and your sample is pretty clean, this may also have an impact on sensitivity.

I think that by "proper SIM" you referred to the so-called t-SIM (targeted-SIM).

[lmh]

yes, that's exactly what I meant by "proper" SIM! I should use the correct Thermo terminology...