Spike of heavy peptides

Spike of heavy peptides nicolas pierre  2021-04-21


We develop a new SRM method with the help of crude heavy peptides. We started with 414 heavy peptides and after refinement we follow 244 heavy peptides. However, our mix of heavy peptides still contains 414 heavy peptides and we found that those peptides spiked at 500 fm each are intense (much more than the light) and decrease the peak quality of the light peptides (in general in create noise). We determined this effect by testing our method with and without the spike of heavy peptides. To ameliorate the quality of our signal, we want to do another mix of heavy peptides only with peptides that we follow (n=244) and we could reduce the quantity of peptides spiked. However, we do not know how to adapt the quantity of heavy peptides. We could try to reach an identical intensity for each heavy peptide (we are not dealing with absolute quantification) but in this case we do not know what is the ideal intensity. Alternatively, we could try to reach an intensity according to the light peptide (1/5, /10 ?). We do not want to have a ratio light/heavy of 1 since at this stage of development we still have some peptides with low intensity and bad quality of peak (refinement is not totally finished, for that we want the best signal quality).
Do you have some advises for this spike of heavy peptides ?

Thank your for your help,

Nicolas Pierre

Brendan MacLean responded:  2021-04-26

Hi Nicolas,
A colleague who sets up mass spectrometry experiments with stable isotope-labeled peptides suggested the following:

"It is a good idea to adjust the amount of heavy peptide to the amount of the endogenous peptide. Ideally you should have a ratio light to heavy close to 1 but if the intensity of the endogenous peptide is too low then an approximate ratio light to heavy 1/10 should work. In this way you ensure the heavy peptide has a good signal (if it doesn't, then the endogenous peptide will probably not be detected) and you don't need to bother about the small isotopic impurities of the heavy peptide."

Hope that helps a little. Good luck with your experiments.


Brendan MacLean responded:  2021-04-26

More feedback from another collaborator with experience setting up these kinds of mixes:

Most logic/beneficial would be to adjust the synthetic peptide concentrations to the endogenous peptide concentrations (ideally a 1:1 ratio). However, you probably will measure your target peptides not only in one sample, but over a series of samples where endogenous concentrations will most likely change (at least that’s usually the reason why peptides were selected initially ;)). So if you adjust the heavy spike-in concentrations to a 1:1 ratio in one sample, this will most likely not be 1:1 ratio in other samples anyway."

Two things tried with relative success:

  1. Generated 2 or 3 endogenous concentration categories, for example high abundant, medium abundant and low abundant. Ideally do this adjustment in a “representative” sample or in a mixed sample from all samples, or a mixed sample from the two most extreme conditions, etc. And then just roughly adjusted the concentrations of the synthetic peptides in the synthetic mix according to these pre-defined categories, but not for each peptide individually.

  2. Trying to reach a rough 1:1 ratio of heavy:light makes sense, but at the same time also make sure that the data quality of the low abundant synthetic peptides are still of decent quality, i.e. do not spike in the low intense peptides in a too low concentration so that you run into data quality issues…