PRMs or SRMs - You decide.

Posted on 27th April, 2020

Informal chat comparing PRMs on TripleTOF to SRMs using a triple quad

 

On the first Tuesday of each month, two proteomics analysts, Pete and Dud, meet up over a few pints to discuss life, the universe and everything. This invariably includes a discussion about mass spectrometry such as this recent chat last month…….

Pete: Good to see you again Dud. 

Dud: You too mate. How’s work going?

Pete: Really good thanks. I’m using a very sensitive triple quad to do some peptide quant work on plasma samples at the moment. Tough matrix, but a great instrument. You just can’t beat a triple in SRM mode for absolute quant work.

Dud: Well, that’s one opinion. I’m sure most people would agree with you. But guess what….

Pete: Don’t tell me. You disagree??

Dud: Afraid so mate. Triples are great and have traditionally been the mass spec of choice for this type of work, but you need to get up to speed with what other options there are.

Pete: Such as?

Dud: Well, have you heard of Parallel Reaction Monitoring, or PRMs ? High-end Q-TOFs like Sciex’s 6600 are more than a match for the triples nowadays.

Pete: OK, I’m listening. But you’ll have your work cut out to convince me to switch from SRMs on a triple. I need to monitor three transitions per peptide, three peptides per protein and I’m looking at twenty proteins simultaneously (all times two as I have the corresponding heavy labelled standards too). That’s a total of 360 separate transitions in a really complex background. Ideally I want this doing in a 30min separation space. What do you think of that?

Dud: Certainly sounds challenging. But what if you could simultaneously monitor ALL of the transitions for each of your peptides with high mass accuracy. In a complex background such as plasma, your selectivity rockets up.

Pete: OK, you’ve got my interest. Selectivity is a major requirement, but I’m looking at very low abundance proteins so sensitivity is key. You can’t touch a triple for sensitivity can you? What do you propose?

Dud: Whilst it is true that the absolute limit of detection is superior with a fully optimised SRM on a triple, you would be surprised how close a high resolution PRM gets to a good old fashioned SRM, without all method development overhead the SRM involves. In most ‘real world’ situations, if your SRM can detect it, the PRM can too, at 10% of the effort. Moreover, the selectivity advantage of multiple transitions (and remember you don’t need to decide which ones you are going to use because you have them all at no additional cost) boosts confidence and reduces false discoveries.

Pete: The selectivity advantage is certainly very appealing. But if I tune each of my transitions to optimal collision energies and voltage settings, then I’m confident that I can’t do any better than that with other instrument formats. With spiked-in heavy-labelled peptides to confirm the elution profile, my selectivity is not exactly shoddy either.

Dud: True, but you said you wanted to monitor 360 separate transitions. How long is that going to take you to set-up? And what do you do if you decide to go after more peptides next time? Your set-up time becomes pretty unjustifiable – especially if you are then only going to analyse a limited number of samples.

Pete: OK. So how long does it take you to set-up the PRM analysis?

Dud: That’s the beauty of method development for PRMs. I don’t have to pre-select the transitions before I acquire the data. They all come for free on a Q-TOF. Just need the precursor mass and a retention time window. One chromatographic run with my synthetic peptides and I’m away. After the data has been acquired I can pull-out my transitions using extracted ion chromatograms (XIC) – with whatever XIC window I like. The tighter the window, the higher the selectivity. Pretty important if plasma is your matrix!

Pete: Mmmm, maybe there is something in this PRM business of interest after all. I guess it’s the usually case of horse for courses in mass spectrometry.

Dud: Come down to the lab and have a bash. By the way, have you got any triple time I could bag, I have this one really tricky low abundance peptide I need to find………………do you want another pint?

 

FEEDBACK

 

Despite having consumed a number of pints of ale, Pete and Dud have presented a number of points relating to merits of PRMs on a TripleTOF versus SRMs on a Triple Quad. Now why not have your say?

 

Who do you agree with the most, PETE or DUD?

 

 

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