Doubly Protonated Species Collision Induced Dissociation for Identification of Isocyclosporins

Abstract

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus <i>Tolypocladium inflatum</i>, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by <i>Tolypocladium inflatum </i>strains of marine origin (White Sea). Cyclosporins A–C contain an unusual amino acid, (4<i>R</i>)-4-((<i>E</i>)-2-butenyl)-4,<i>N</i>-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporine by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]<sup>+</sup> ions due to the rapid [CsA+H]<sup>+</sup>→[isoCsA+H]<sup>+</sup> conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]<sup>2+</sup> ions, and their MS/MS fragmentation can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. The fragmentation patterns of [CyA+2H]<sup>2+</sup> and [isoCyA+2H]<sup>2+</sup> ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.