Maurits A. den Boer

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of─among others─biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein-ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

Significance Antibodies are crucial for the immune response against bacteria. To drive bacterial killing, antibodies should bind to the bacterial cell and induce the complement reaction. This requires target-bound IgGs to form hexameric IgG platforms that are kept together by noncovalent Fc-Fc interactions. Interestingly, pathogenic bacteria produce IgG-binding molecules that bind specifically to the Fc region needed for hexamerization. Here we demonstrate that staphylococcal protein A (SpA) from Staphylococcus aureus specifically blocks formation of IgG hexamers and downstream activation of complement. Furthermore, we show that IgG3 antibodies (which are not recognized by SpA) have superior capacity to activate complement and induce killing of S. aureus by human phagocytes. These insights provide a crucial rationale for optimizing antibody therapies against S. aureus .

BACKGROUND: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. METHODS: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). RESULTS: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. CONCLUSIONS: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Qualitative and quantitative mass analysis of antibodies and related macromolecular immune complexes is a prerequisite for determining their identity, binding partners, stoichiometries, and affinities. A plethora of bioanalytical technologies exist to determine such characteristics, typically based on size, interaction with functionalized surfaces, light scattering, or direct mass measurements. While these methods are highly complementary, they also exhibit unique strengths and weaknesses. Here, we benchmark mass photometry (MP), a recently introduced technology for mass measurement, against native mass spectrometry (MS) and size exclusion chromatography multi-angle light scattering (SEC-MALS). We examine samples of variable complexity, namely, IgG4Δhinge dimerizing half-bodies, IgG-RGY hexamers, heterogeneously glycosylated IgG:sEGFR antibody-antigen complexes, and finally megadalton assemblies involved in complement activation. We thereby assess the ability to determine (1) binding affinities and stoichiometries, (2) accurate masses, for extensively glycosylated species, and (3) assembly pathways of large heterogeneous immune complexes. We find that MP provides a sensitive approach for characterizing antibodies and stable assemblies, with dissociation correction enabling us to expand the measurable affinity range. In terms of mass resolution and accuracy, native MS performs the best but is occasionally hampered by artifacts induced by electrospray ionization, and its resolving power diminishes when analyzing extensively glycosylated proteins. In the latter cases, MP performs well, but single-particle charge detection MS can also be useful in this respect, measuring masses of heterogeneous assemblies even more accurately. Both methods perform well compared to SEC-MALS, still being the most established method in biopharma. Together, our data highlight the complementarity of these approaches, each having its unique strengths and weaknesses.