Hello all,
I am setting up a method to relatively quantify several proteins of interest on the Q-Exactive. I am in doubt of which method to use.
The first option is t-SIM with a dd-MS2 for peak identification. The other option is PRM (t-MS2). In the first option, quantitation will be performed on MS1 data. In the second option, quantitation will be performed on MS2 data.
Which is best and why?
I would use tsimddmsms and use MS1 ions (parent) for quantitation because they would be more sensitive than MS2 ions. Apart from that MS/Ms can be set to be data(intensity, incl list) dependent in this mode, but not in tms/ms.
I had the same qn while working with q exactive and have discussed about these modes in the article attached
Article Targeted analysis with benchtop quadrupole-orbitrap hybrid m...
Depends on your background matrix, technically your signal to noise will be increased with t-ms2 data and you gain selectivity. From my experience, a Full-MS scan (at high res) followed by targeted-ms2 scans with inclusion list will work good enough. That way you can use the survey scan at high res and overlay them over the fragments. This also allows you to use isotopic distribution of the parent ion and select the correct ms2 fragments from the interference. But still depends on your sample complexity and dynamic range so you should do a test.
I would not recommend to perform SIM or MS/MS analysis in a QExactive for quantification purposes, as your MS spectrum working in the full scan mode will be good enough to do so. If you want to have the MS/MS spectrum at the same time, I would recommend target MS/MS with a bigger window in the quadrupole (for ex. 4Da , rather than 1 Da...). However, there has been a huge increase in the number of publications (especially in the International Mass Spectrometry Conference held at Geneva last summer) working with DIA (Data Independent Acquisition). Seems to work fine in the proteomics field... Here you can find a vast number of publications: http://scholar.google.es/scholar?q=Data+Independent+Acquisition&hl=es&as_sdt=0&as_vis=1&lookup=0&oi=scholart&sa=X&ei=mvgyVOPiKKTP7QaFkYCACA&ved=0CCMQgQMwAA.
Nevertheless, quantification should be performed with the full scan or SIM...
Good luck!
Thank you all for your answers so far. What I should have mentioned in the original question is that I want to measure as sensitive as possible. My background matrix is relatively clean. Samples are obtained from in-gel digestion of specific protein bands, so I am expecting relatively pure samples.
What I understand from your answers so far is that the best way to do it is to quantify on the MS1 level. So this could either be a full MS scan or a SIM. What would be the added benefit of doing full scans? It feels like you are doing a lot of redundancy that way and I am afraid I will lose signal, and thus sensitivity, that way. Since I know the m/z and retention times of my peptides of interest, I would say that a targeted SIM approach may be best, wouldn't you agree?
Hi Alexander,
I'd work in the full scan mode and then extract your ions of interest. You won't win signal if you work in the sim mode and then you'd have all the MS spectra, that's the magic of the orbitrap. It's an ion trap, so the background noise it's not very high, as it happens with the quadrupoles.
Also I'd recommend skyline software: https://skyline.gs.washington.edu/labkey/wiki/home/software/Skyline/page.view?name=default. It's highly used by proteomics people!
For targeted and sensitive analysis, tSIM or tSIM dd MSMS mode would be better.. In full scan mode, you would notice the c trap suppression of your analyte because the ctrap will be filled with your analyte + matrix ions. In full scan mode, more the matrix in your sample, more the suppression of your anlayte signal..On the other hand in tSIM, Ctrap will contain only quadrupole filtered analyte ions and hence sensitive analyte signal!
Dear Alexander,
the answer is - it depends. I would like to add a few comments: on Orbitrap analyzers tSIM is significantly more sensitive than full scan analysis (Galien et al., Mol Cell Proteomics. 2012 Dec;11(12):1709-23.) so no matter what you do, targeted analysis is a good idea to get to highest sensitivity. As to the choice between tSIM and PRM, it depends on your background. PRM is MS/MS based so has inherently higher specificity, however the overall "base" sensitivity is lower as you distribute the available precursor ions across multiple fragments. PRM usually wins over tSIM if your matrix is highly complex as it works by improving signal-to-noise rather than absolute signal. If your matrix is only moderately complex as you state, tSIM is often the better option as the absolute signal is the limiting factor here.
The best way is to actually try out both approaches using a quick series of spike dilutions if you have standards in hand. It is done in a day and will show what works better.
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