Structural Characterization of Daunomycin-Peptide Conjugates by Various Tandem Mass Spectrometric Techniques.

Ildikó Szabó,Mohammed Al-Majidi,Tibor Nagy,Gitta Schlosser,Adina Borbély,Sándor Kéki,Gergő Kalló,Gábor Mező,Lilla Pethő,Éva Csősz,Arnold Steckel

doi:10.3390/ijms22041648

Abstract

The use of peptide-drug conjugates has generated wide interest as targeted antitumor therapeutics. The anthracycline antibiotic, daunomycin, is a widely used anticancer agent and it is often conjugated to different tumor homing peptides. However, comprehensive analytical characterization of these conjugates via tandem mass spectrometry (MS/MS) is challenging due to the lability of the O-glycosidic bond and the appearance of MS/MS fragment ions with little structural information. Therefore, we aimed to investigate the optimal fragmentation conditions that suppress the prevalent dissociation of the anthracycline drug and provide good sequence coverage. In this study, we comprehensively compared the performance of common fragmentation techniques, such as higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron-transfer higher energy collisional dissociation (EThcD) and matrix-assisted laser desorption/ionization–tandem time-of-flight (MALDI-TOF/TOF) activation methods for the structural identification of synthetic daunomycin-peptide conjugates by high-resolution tandem mass spectrometry. Our results showed that peptide backbone fragmentation was inhibited by applying electron-based dissociation methods to conjugates, most possibly due to the “electron predator” effect of the daunomycin. We found that efficient HCD fragmentation was largely influenced by several factors, such as amino acid sequences, charge states and HCD energy. High energy HCD and MALDI-TOF/TOF combined with collision induced dissociation (CID) mode are the methods of choice to unambiguously assign the sequence, localize different conjugation sites and differentiate conjugate isomers.

Highlights

In recent years, peptide-drug conjugates (PDCs) have become an effective approach for the targeted delivery of therapeutic agents [1]
Four α-MSH-daunomycin bioconjugates were chosen as model compounds to inves2.1
Synthesis tigate the most optimal activation technique for the structural characterization of daunoFour α-MSH-daunomycin were chosen as model to inmycin-based bioconjugates and bioconjugates to illustrate possible differences in compounds

Summary

Introduction

Peptide-drug conjugates (PDCs) have become an effective approach for the targeted delivery of therapeutic agents [1]. In this strategy one or more chemotherapeutic agents are covalently attached to peptides which can serve as carriers to selectively deliver cytotoxic drugs to solid malignancies. Given that α-MSH peptide analogs bind to melanocortin-1 (MC1) receptors, they are promising drug carriers in melanoma therapy [5]. The analog containing Nle at position 4, as well as the central fragments containing lysine residues exhibit high melanotropic activity, providing a wide platform for PDC development with different sites for drug conjugation [6]

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Conclusion