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PRM reproducibility question jung165  2018-02-13 19:38
 
Hello this is Jinwoo Jung who are working with PRM analysis.

I am about to start PRM analysis for my experiment and I have one quick question about its reproducibility

I have performed PRM analysis with Q-exactive device with triplicate run but it shows some peptides with over 30% coefficient variance between multiple analysis.

Retention time variance is about 1~2 minutes. I think this is not much acceptable values for variance.
I also analyzed same peptide with SRM, but it shows almost same retention time between multiplicate run.

I would like to ask those variances are acceptable. If not, is there any parameters that need to be modified for better result???

I also attached my result and method of PRM analysis

I really appreciate for your help.
 
 
Brendan MacLean responded:  2018-02-14 11:59
Hi Jung,
That is not great RT reproducibility. If those runs were don consecutively, then you should definitely look at what you are doing for chromatography. Perhaps your column is not fully conditioned for your sample on the first run?

In the field, <20% CV, especially for just 3 replicates, is considered acceptable, but your median across many peptides should definitely be considerably lower. So 30% is not good.

I would probably try running more replicates. Look at your chromatography for sure.

In the one set of chromatograms you show, it appears to me that the transitions you have chosen are experiencing interference on either side of your target peptide. Look at the shoulders (bumps) on either side that occur on only some of the transitions.

So, it seems there may be multiple reasons for your poor reproducibility. It should be possible to get much higher reproducibility out of PRM on a Q Exactive instrument.

Good luck troubleshooting your issues. Thanks for posting to the Skyline support board.

--Brendan