Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human Neutrophil Peptides (HNPs)

Antimicrobial peptides (AMPs) are known best for their role in innate immunity against bacteria, viruses, parasites and fungi. However, not only are they showing increasing promise as potential antimicrobial drug candidates, recently, it has been reported that certain AMPs also show a cytotoxic effect against cancer cells. Their possible antitumor effect could make AMPs interesting candidate cancer biomarkers and a possible lead for new anticancer therapy. Due to their cyclic structure, detection and identification of AMPs is challenging, however, mass spectrometry (imaging; MSI) has been shown as a powerful tool for visualization and identification of (unknown) cyclic AMPs. In this chapter, we will discuss how mass spectrometry (imaging), combined with the use of electron-transfer dissociation (ETD) as fragmentation technique, can be used as a reliable method to identify AMPs in their native cyclic state. Using this approach, we have previously detected and identified human neutrophil peptides (HNPs) as important AMPs in cancer, of which a detailed bacterial, viral and cancer-related overview will be presented.

O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

Profiling Dopamine-Induced Oxidized Proteoforms of β-Synuclein by Top-Down Mass Spectrometry

The formation of multiple proteoforms by post-translational modifications (PTMs) enables a single protein to acquire distinct functional roles in its biological context. Oxidation of methionine residues (Met) is a common PTM, involved in physiological (e.g., signaling) and pathological (e.g., oxidative stress) states. This PTM typically maps at multiple protein sites, generating a heterogeneous population of proteoforms with specific biophysical and biochemical properties. The identification and quantitation of the variety of oxidized proteoforms originated under a given condition is required to assess the exact molecular nature of the species responsible for the process under investigation. In this work, the binding and oxidation of human β-synuclein (BS) by dopamine (DA) has been explored. Native mass spectrometry (MS) has been employed to analyze the interaction of BS with DA. In a second step, top-down fragmentation of the intact protein from denaturing conditions has been performed to identify and quantify the distinct proteoforms generated by DA-induced oxidation. The analysis of isobaric proteoforms is approached by a combination of electron-transfer dissociation (ETD) at each extent of modification, quantitation of methionine-containing fragments and combinatorial analysis of the fragmentation products by multiple linear regression. This procedure represents a promising approach to systematic assessment of proteoforms variety and their relative abundance. The method can be adapted, in principle, to any protein containing any number of methionine residues, allowing for a full structural characterization of the protein oxidation states.

Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.