Absolute quantification of cell surface proteins and their complexes

Xu, Shuhan (2026): Absolute quantification of cell surface proteins and their complexes. Dissertation, LMU München: Faculty of Biology

Abstract

The advancement of super-resolution fluorescence microscopy has enabled the study of subcellular structures at the single-protein level, particularly at the cell surface, where the spatial organization of signaling proteins can be explored. However, quantifying the absolute copy number of cell surface proteins and their complexes requires a general method to evaluate the labeling efficiency of molecular binders, which remains unavailable. In this thesis, I developed a simple and broadly applicable approach to determine the labeling efficiency of binders, allowing the selection of optimal reagents, assessment of conjugation methods, and quantitative analysis of the fraction of proteins that form larger complexes. To accelerate binder characterization, I established a faster, diffraction-limited imaging approach for estimating labeling efficiency without requiring chemical modification of the binders. This method enables rapid binder screening and highlights the importance of using monoclonal, site-specifically labeled secondary binders to ensure consistent and quantitative results. I also investigated the generation and labeling of antibody-derived Fab fragments as potential reagents for single-molecule imaging. While antibodies are widely available, their large size can introduce labeling uncertainty and their bivalency can limit quantitative accuracy. I found that some Fab fragments exhibited reduced labeling efficiency and increased nonspecific binding compared to their parent antibodies, likely due to changes to the structure or physical properties of the binders. Finally, I applied a simulation and modeling framework that incorporates binder properties, including labeling efficiency, to quantify the proportion of proteins in complexes. As a proof of principle, I used this method to quantify CD80-PD-L1 interaction. This approach could be used to assess how PD-L1 availability on antigen-presenting cells can contributes to T-cell regulation in future studies. Collectively, the methods developed in this work enhance quantitative fluorescence microscopy by enabling accurate analysis of protein labeling and interactions, advancing our understanding of cell signaling mechanisms.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 5. August 2026
• 1. Referee: Jungmann, Ralf
• Signature of the printed copy: 0001/UMC 32240