“MS/MS Methods for Explicit Determination of Bioactive Proteforms”
Professor Catherine Costello, Boston University, School of Medicine
Increased understanding of infection, cancer, and the immune system is providing us with powerful glycoprotein-based drugs and diagnostic tools. Infectious agents usually gain entrance to their hosts through the interactions of surface molecules. The immune system is responsible for and exploits the interactions of proteins with one another and with glycans (and other classes). In order to explore these phenomena, to investigate how the body can combat challenges, yet its response may itself have deleterious consequences, and to utilize this knowledge to control disease, we now rely heavily on the tools provided by mass spectrometry. This lecture will review some of the types of inter- and intra-molecular interactions that are important in infection, carcinogenesis, and neurodegeneration and the mass spectrometry approaches that we are using and developing further to elucidate the critical players in these pathways.
“PepSAVI-MS Reveals Anticancer and Antifungal Cycloviolacins in Viola odorata”
Nicole Parsley, Laboratory of Professor Leslie Hicks, University of North Carolina-Chapel Hill, Department of Chemistry
Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized Via Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature’s complexity. Using PepSAVI-MS, we aim to characterize these novel cyclotides from V. odorata via isolation, sequence and structure characterization using LC-MS/MS and NMR techniques, and bioactivity determination by screening against fungal pathogens.