Complete Sequencing of a Hybrid Peptide/Peptide-Nucleic Acid Construct with MALDI-TOF/TOF Mass Spectrometry

Peptide-nucleic acids (PNAs) remain a promising therapeutic modality, although their success in the clinic remains modest compared to other nucleic acid-based products, such as thiophosphoryl and thiophosphoramide oligonucleotides. To address this, extensive efforts have been made in the past decade to optimize the structure of PNAs aiming at improving their manufacturability, in vivo stability, specificity and targeted delivery. Large sizes and complex structures of the newest-generation PNAs (featuring nonuniform distribution of backbone modifications to increase stability and enhance binding to therapeutic targets, and incorporating extended peptide segments serving as targeted delivery vectors) pose unique challenges vis-à-vis structural characterization of these molecular entities. We use a top-down MS/MS approach to sequence a 6 kDa hybrid PNA molecule comprising a short polybasic cell permeabilizing segment (CPS) and a long antisense sequence segment, and containing γ-hydroxymethyl groups distributed unevenly across the backbone to stabilize the helical structure. Localization of the positive charge within the CPS enables backbone cleavages only within this PNA segment when conventional ion fragmentation methods are used, while the non-natural γ-peptide backbone prevents the use of proteases to assist the structural analyses. However, high-energy collisional activation (implemented with MALDI TOF/TOF) provides complete sequence coverage of the entire PNA molecule, including localization of all γ-hydroxymethyl groups, thereby enabling de novo sequencing of this molecular entity. The use of the fragment ions mass spectrum of the reference PNA molecule enables "mass fingerprinting" analysis by allowing the isomeric PNA molecules with scrambled sequences to be readily distinguished from the reference PNA.