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.