Tandem mass spectrometry (MSn) of peptide disulfide regio-isomers via collision-induced dissociation: Utility and limits in disulfide bond characterization

Abstract

Mass spectrometric characterization of the disulfide connecting patterns directly from intact peptides and proteins is highly desirable but remains a challenging task. In this work, the regio-isomers of peptides containing two intrachain disulfide bonds were synthesized from P1 and P2 peptides (single letter sequence: C(1)ARIC(5)AKLC(9)LEVC(13)K and C(1)AEKC(5)IEKC(9)LVRC(13), respectively). They were further used as model systems to understand the fragmentation chemistry of each isomer under low energy collision-induced dissociation (CID) conditions. MS2 CID could easily identify the regio-isomer having a side-by-side disulfide linkage pattern (C1–C5 and C9–C13). However, the other two isomers with either loop-within-a-loop (C1–C13 and C5–C9) or overlapped disulfide configuration (C1–C9 and C5–C13) showed almost identical spectra and very limited sequence information could be obtained. Internal fragments which resulted from cleavages of two amide bonds from a sequence covered only by one disulfide loop were chosen for further dissociation. The MS3 CID data showed that certain internal fragment ions produced distinct fragmentation patterns which were useful in assigning the correct connecting pattern of the disulfide bonds for all three isomers. On the other hand, some internal fragment ions could undergo consecutive disulfide bond opening during collisional activation, which led to the observation of isomeric peaks from different disulfide regio-isomers. The latter situation made it difficult to independently assign the regio-isomers.