POSTER SESSION ABSTRACTS

Peak assignments for the MS/MS mass spectra of cyclic peptides with multiple disulfide bridges are a challenge to analyze. In addition to the standard peptide cleavages, each disulfide linkage may undergo up to four possible fragmentation processes. The MASSPEC algorithm was designed to exhaustively compute all possible substructures of ions consistent with mass spectral fragmentation rules. The MASSPEC algorithm correlates the predicted substructure masses with observed fragment ion masses and scores all assignments. In this way, the MASSPEC algorithm is a powerful tool for the elucidation of the complex fragmentation processes observed in the MS/MS spectra of disulfide-linked cyclic peptides. The data for disulfide-linked peptides and related metabolites were acquired using a ThermoFisher Q Exactive Orbitrap mass spectrometer in the LC-MS/MS mode of the [M+H] parent ion under stepped collision energy (RP = 35,000, D = 3 ppm). MS experiments were conducted on an LTQ-Orbitrap. The MASSPEC algorithm is written in Visual Basic and is based upon advanced graph theory and combinatorial methods. User interface features used for the assays include a Data Input Module and an Output Graphics Display Module. Using the MASSPEC algorithm, the proposed structures for the fragment ions were obtained automatically by computing the highest scoring fragment ion substructures for the observed ions. The MASPEC algorithm was applied to MS/MS data in the following manner. One and/or three superatom models were used to describe the peptide residues while the disufide linkages were re-expressed in superatom notation as C'-S'-HH-S'-C', where C' represents dehydroalanine, S' represents sulfur and HH represents two hydrogen atoms. The cleavage of the C'-S' bonds can result in the formation of persulfide and dehydroalanine groups and cleavage of the S'-HH bonds can result in the formation of cysteine and thialdehyde groups. In this manner, model grafted cyclotides (cyclic peptides with three disulfide bridges) were analyzed. Orbitrap ESI-LC-MS/MS data for the parent grafted cyclotide and corresponding metabolites were acquired and evaluated. The MASSPEC Hydrolyzer Algorithm was used to generate all the possible metabolite structures for each parent compound. The highest scoring structure for the metabolite MS/MS data corresponded to the correct metabolite when checked against enzymatic data.