Emerging mass spectrometry-based approaches to probe protein-receptor interactions: focus on overcoming physiological barriers

Abstract

Physiological barriers, such as the blood-brain barrier and intestinal epithelial barrier, remain significant obstacles towards wider utilization of biopharmaceutical products. Receptor-mediated transcytosis has long been viewed as an attractive means of crossing such barriers, but successful exploitation of this route requires better understanding of the interactions between the receptors and protein-based therapeutics. Detailed characterization of such processes at the molecular level is challenging due to the very large physical size and heterogeneity of these species, which makes use of many state-of-the art analytical techniques, such as high-resolution NMR and X-ray crystallography impractical. Mass spectrometry has emerged in the past decade as a powerful tool to study protein-receptor interactions, although its applications to investigate interaction of biopharmaceuticals with their physiological partners are still limited. We highlight the potential of this technique by considering several recent examples where it had been instrumental for understanding molecular mechanisms critical for receptor-mediated transcytosis of transferrin-based therapeutics.

Keywords: BBB; Blood–brain barrier; CNS; CSF; ESI; Fusion proteins; GHT; Growth hormone; HDX; Intestinal epithelial barrier; Lysozyme; Lz; LzT; MS; Protein aggregation; Protein therapeutics; Protein–drug conjugate; Receptor-mediated transcytosis; Tf; TfR; Transferrin; Transferrin receptor; central nervous system; cerebrospinal fluid; electrospray ionization; growth hormone-transferrin fusion protein; hydrogen/deuterium exchange; lysozyme; lysozyme–transferrin conjugate; mass spectrometry; transferrin; transferrin receptor.

Figure 1

A schematic diagram representing the blood-brain barrier (top), and the transport of proteins from luminal side of BBB (capillary) to abluminal side (brain) via receptor mediated endocytosis. Adopted from (83).

Figure 2

Schematic depiction of the intestinal epithelium and the pathways available for drug absorption: (a) transcellular pathway through the epithelial cells; (b) paracellular pathway (in between adjacent cells), only small hydrophilic molecules are absorbed through this pathway, and even in these cases the absorption is quite limited because the paracellular pathway comprises a very small percentage of the total epithelial surface area; (c) transcytosis and receptor-mediated endocytosis; and (d) absorption into the lymphatic circulation via M-cells of Peyer’s patches. Adapted with permission from (84).

Figure 3

Schematic representation of targeted delivery of Tf-based therapeutic agents to intracellular targets using TfR-mediated endocytosis (a) and across a physiological barrier using TfR-mediated transcytosis (b).

Figure 4

Localization of the receptor binding interface within diferric Tf using HDX MS. The panels show isotopic distributions of representative peptic fragments derived from the protein subjected to HDX in the presence (blue) and the absence (red) of the receptor. The black traces at the bottom of each diagram show isotopic distributions of peptic fragments derived from unlabeled protein, and the dotted lines represent the end-points of the exchange reaction. Colored segments within the Tf/TfR complex show location of the peptic fragments. Adapted with permission from (85).

Figure 5

ESI mass spectra of Tf/TfR (top) and LzT/TfR (bottom) mixtures acquired under near-native conditions (3 μM of each protein in 20 mM ammonium acetate at pH 7.1). Peak labels represent the charge states of all relevant ions. The inset shows a mass spectrum of LzT spiked with intact Tf. Adapted with permission from (70).

Figure 6

SEC of GHT oligomers, TfR and their mixture (A), and total ion chromatograms (B) of tryptic digests of the SEC fractions of TfR (blue trace) and the TfR/GHT oligomer mixture (red trace) highlighted in panel A. Panels C and D illustrate detection of TfR in the early-eluting SEC fraction of GHTx/TfR mixture (highlighted with red in panel A): single-scan MS/MS spectrum (C) and extracted ion chromatogram (D) of a tryptic peptide eluting at 25 min (marked with asterisk in panel B). The blue trace in panel C shows the reference MS/MS spectrum of a tryptic fragment T71 (LTTDFNAEK) of TfR, and the blue trace in panel D corresponds to this peptide derived from TfR in the absence of GHT oligomers (SEC fraction highlighted with blue in panel A). Adapted with permission from (20).