Regl, C. | bioRxiv Science

bioRxiv2025

A new class of proteinaceous biomaterial with unprecedented properties in unicellular eukaryotes

Biomaterials fundamentally reshape material use, providing superior properties and sustainable alternatives relevant to medicine, textiles, and high-tech applications. Research mainly focuses on animal-derived proteinaceous biomaterials, which remain challenging to reproduce while retaining their remarkable properties. We discovered that the shells of tintinnid ciliates, a group of planktonic unicellular eukaryotes, are composed of self-assembling structural proteins. The shells form in water, are structurally diverse, and show exceptional resistance against high temperatures and the strongest chemicals. Combining single-cell transcriptomics and mass-spectrometry of the shells, we identified the amino acid sequences of the shell-forming proteins. They are exceptionally rich in aromatic residues and possess a unique architecture with flexible, unfolded segments connecting highly stable beta-sheets. This easily accessible system promises new aspects to advance current biomaterial design.

molecular biology↗

bioRxiv2020

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

Advanced analytical strategies including top-down and middle-up HPLC-MS approaches have become powerful alternatives to classical bottom-up analysis for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up analysis of polyclonal IgGs posing additional challenges due to extensive sequence variability. The presented workflow is based on Fc/2 portions as conserved subunits of IgGs and enables global profiling of subclasses and their glycosylation patterns, both of which influence IgG effector functions. To obtain subunits of murine IgGs, we established digestion with the bacterial protease SpeB. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry allowing relative quantification of IgG subclasses and their N-glycosylation variants. In order to assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. This analysis simultaneously revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine as demonstrated for monoclonal IgGs, which may be implemented for quality control of functional antibodies.

biochemistry↗