Grandke, J. | bioRxiv Science

bioRxiv2026

A Scalable and Robust Workflow for Cost-Effective Post-Translational Modifications Profiling by Chemical Proteomics

Mass spectrometry-based chemical proteomics is a powerful method to analyze proteins labelled by small molecules to identify protein targets of active compounds and to profile protein post-translational modifications. The throughput and high protein input for chemical proteomics workflows has been often a limiting factor for application of the technology for specialized and difficult to culture cell lines. The high protein input was necessary to gain significant difference of noise to signal ratio in proteomics readout. Here, we describe a general chemical proteomics workflow, which is performed in 96-well plate and necessitate only 25 g of protein input to profile post-translationally modified proteins including abundant O-GlcNAcylated proteins as well as low abundant AMPylated proteins. The workflow integrates advances in Cu(I)-catalyzed azide-alkyne cycloaddition to minimize chemical side-reactivity of the click reaction and data-independent acquisition mode during LC-MS/MS measurement. An iterative optimization of protein clean-up on carboxylate-coated paramagnetic beads led to significant saving of the beads usage and lowers the unspecific protein background that resulted in sensitivity gain.

biochemistry↗

bioRxiv2019

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Youngs modulus E) within the physiologically relevant range from 1 kPa to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

biophysics↗