Overcharging Effect in Electrospray Ionization Mass Spectra of Daunomycin-Tuftsin Bioconjugates

Abstract

Peptide-based small molecule drug conjugates for targeted tumor therapy are currently in the focus of intensive research. Anthracyclines, like daunomycin, are commonly used anticancer drug molecules and are also often applied in peptide-drug conjugates. However, lability of the O-glycosidic bond during electrospray ionization mass spectrometric analysis hinders the analytical characterization of the constructs. “Overprotonation” can occur if daunomycin is linked to positively charged peptide carriers, like tuftsin derivatives. In these molecules, the high number of positive charges enhances the in-source fragmentation significantly, leading to complex mass spectra composed of mainly fragment ions. Therefore, we investigated different novel tuftsin-daunomycin conjugates to find an appropriate condition for mass spectrometric detection. Our results showed that shifting the charge states to lower charges helped to keep ions intact. In this way, a clear spectrum could be obtained containing intact protonated molecules only. Shifting of the protonation states to lower charges could be achieved with the use of appropriate neutral volatile buffers and with tuning the ion source parameters.