Mass Spectrometry-Guided Optimization and Characterization of a Biologically Active Transferrin–Lysozyme Model Drug Conjugate

Transferrin is a promising drug carrier that has the potential to deliver metals, small organic molecules and therapeutic proteins to cancer cells and/or across physiological barriers (such as the blood–brain barrier). Despite this promise, very few transferrin-based therapeutics have been developed and reached clinical trials. This modest success record can be explained by the complexity and heterogeneity of protein conjugation products, which also pose great challenges to their analytical characterization. In this work, we use lysozyme conjugated to transferrin as a model therapeutic that targets the central nervous system (where its bacteriostatic properties may be exploited to control infection) and develop analytical protocols based on electrospray ionization mass spectrometry to characterize its structure and interactions with therapeutic targets and physiological partners critical for its successful delivery. Mass spectrometry has already become an indispensable tool facilitating all stages of the protein drug development process, and this work demonstrates the enormous potential of this technique in facilitating the development of a range of therapeutically effective protein–drug conjugates

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography

A new approach for constant-distance mode mass spectrometry imaging (MSI) of biological samples using nanospray desorption electrospray ionization (nano-DESI) was developed by integrating a shear-force probe with the nano-DESI probe. The technical concept and basic instrumental setup, as well as the general operation of the system are described. Mechanical dampening of resonant oscillations due to the presence of shear forces between the probe and the sample surface enabled the constant-distance imaging mode via a computer-controlled closed-feedback loop. The capability of simultaneous chemical and topographic imaging of complex biological samples is demonstrated using living Bacillus subtilis ATCC 49760 colonies on agar plates. The constant-distance mode nano-DESI MSI enabled imaging of many metabolites, including nonribosomal peptides (surfactin, plipastatin, and iturin) on the surface of living bacterial colonies, ranging in diameter from 10 to 13 mm, with height variations up to 0.8 mm above the agar plate. Co-registration of ion images to topographic images provided higher-contrast images. Based on this effort, constant-mode nano-DESI MSI proved to be ideally suited for imaging biological samples of complex topography in their native states