Charge site manipulation to enhance top-down fragmentation efficiency.

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency.

Stable Binding of Full-Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus.

The adipokine chemerin is the endogenous ligand of the chemokine-like receptor 1 (CMKLR1), a member of the family of G protein-coupled receptors (GPCRs). This protein ligand plays an important role in obesity and inflammatory processes. Stable receptor-ligand interactions are highly relevant for its different physiological effects such as the migration of immune cells towards sites of inflammation. Here, we demonstrate that negative charges in the CMKLR1 N terminus are involved in the formation of strong contacts with a specific positively charged patch at the surface of full-length chemerin, which is absent in the short nonapeptide agonist chemerin-9, thus explaining its reduced affinity. Using receptor chimera of G protein-coupled receptor 1 (GPR1) and CMKLR1, we were able to identify the residues of this interaction and its relevance for stable full-length chemerin binding. This could help to develop more potent ligands for the treatment of inflammation-related diseases.

Pinpointing the interaction site between semaphorin-3A and its inhibitory peptide.

Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A-NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l-phenylalanine or photo-l-leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A-NRP-1 interaction.

The Chemerin Receptor CMKLR1 Requires Full-Length Chemerin for High Affinity in Contrast to GPR1 as Demonstrated by a New Nanoluciferase-Based Binding Assay.

To study the binding mode of the adipokine chemerin as well as the short peptide agonist chemerin-9 (C9) to its two receptors chemokine-like receptor 1 (CMKLR1) and G protein-coupled receptor 1 (GPR1), we generated 5-carboxytetramethylrhodamine (TAMRA) modified variants of both ligands. In addition, we labeled GPR1 and CMKLR1 with a nanoluciferase at the N-terminus to perform NanoBRET binding assays. For GPR1, both ligands show high affinity and comparable binding. Significant differences were found for CMKLR1, whereby only full-length chemerin binds with high affinity in saturation and displacement assays. For TAMRA-C9 a biphasic binding consisting of two binding states has been found and no displacement studies could be performed. Thus, we conclude that CMKLR1 requires full-length chemerin for stable binding in contrast to GPR1. This work demonstrates the NanoBRET binding assay as a new tool for binding studies at chemerin receptors and it enables deeper insights into the ligand binding parameters.