Scheme 1.

To illustrate isobaric interference, two co-eluting DiLeu-labeled isobaric peptides, Peptide A (z = +2) and Peptide B (z = +3), are shown being analyzed on a SYNAPT G2 mass spectrometer. Peptide A has an expression ratio of 5:1:5:1 and Peptide B has an expression ratio of 1:5:1:5. (a) If IM separation is disabled, the two peptides will be co-fragmented resulting in an inaccurate reporter spectrum that is not representative of either peptide's true expression ratio. (b) On the other hand, if IM separation is enabled, the precursors will enter the Transfer stack-ring ion guide (SRIG) separately, allowing each peptide to be fragmented and mass analyzed in the TOF individually producing accurate reporter ion expression ratios for each peptide.