Chromatography Forum

I really need some suggestions on this problem, thanks in advance!

I am working on a project to quantify a synthetic peptide in rat plasma using LC/MS MRM technique. Currently I am using two MRM pairs for my target peptide (parent ions are +5 and +6 charge states of the molecular ion and the product ion is same for both transitions) and I did not use any enzymatic digestion (peptide MW is ~3200 and quiet polar). The sequence of this peptide contains two parts (which I will call it A and B, so the peptide is A+B). The "potential" internal standard for this peptide is another peptide whose sequence is A+B' (where B' is the exact reverse sequence of B). So these two peptides have the same molecular weight.

I am concerned whether the internal standard peptide would be applicable in this case since the two MRM pairs that I chose would be the same and the RT of these peptides should also be very similar (if not same). I don't have isotopically labeled peptides. How could this peptide serve as IS for my target peptide? Must I use enzymes to digest them (to seek different MRM pairs between the two peptides) prior to LC/MS?

Any suggestions are highly appreciated.

If your analyte is made up of A+B and the IS is A+B', where B' is attached to A in exactly the reverse order that B is attached to A.... Then I would expect that if you fragmented analyte and IS (MRM) you very well could see different fragment ions and thus different mass transitions for each A+B and A+B'. You may have to optimize the collision energy.