Hello,
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