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

•Peptide disulfide regio-isomers containing two intrachain disulfide bonds were synthesized and characterized via tandem mass spectrometry.•The disulfide bond connecting patterns can be determined based on MS3 CID of the internal ion losses derived from each isomer.•Consecutive disulfide bond opening during MS3 CID of the internal loss ions can produce isomeric fragment ions, making independent disulfide assignment difficult.

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.

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