LC-Trapped Ion Mobility Spectrometry-TOF MS Differentiation of 2-and 3-Disulfide-Bonded Isomers of the μ-Conotoxin PIIIA

Disulfide bonds within cysteine-rich peptides are important for their stability and biological function. In this respect, the correct disulfide connectivity plays a decisive role. The differentiation of individual disulfide-bonded isomers by traditional high-performance liquid chromatography (HPLC) and mass spectrometry (MS) is limited due to the similarity in physicochemical properties of the isomers sharing the same amino acid sequence. By using trapped ion mobility spectrome-try-mass spectrometry (TIMS-MS), several 2- and 3-disulfide-bonded isomers of the mu-conotoxin PIIIA were investigated for their distinguishability by collision cross section (CCS) values and their characteristic mobilogram traces. The isomers could be differentiated by TIMS-MS and also identified in mixing experiments. Thus, TIMS-MS provides a highly valuable and enriching addition to standard HPLC and MS analysis of conformational isomers of disulfide-rich peptides and proteins.