Elucidating protease substrate specificities with enhanced secretome profiling techniques

The secretome is crucial for many biological processes, including development and neurodegeneration. Mass spectrometry-based proteomics offers profound insights into the cellular secretome, highlighting quantitative changes in disease contexts. To enhance secretome analysis, our group implemented the High-performance Secretome Protein Enrichment With Click Sugars (hiSPECS) method, incorporating lectin-based glycoprotein enrichment, direct azide glycoprotein coupling to alkyne beads, and on-bead proteolysis (Tushaus et al., 2020). Now, we further improved sensitivity of our method through the use of advanced magnetic beads and automated washing steps.

Proteolytic cleavage of membrane proteins, known as shedding, releases ectodomains that become integral components of the secretome. Therefore, secretome analytics is ideally suited to identify physiologically relevant substrates of membrane proteases like ADAM10. ADAM10, a widely expressed transmembrane protein, functions as a "molecular scissor," mediating ectodomain shedding by cleaving extracellular domains from membrane protein substrates. With over 100 substrates, including Notch, amyloid precursor protein, cadherins, and growth factors, ADAM10 is crucial in both health and disease, particularly cancer and Alzheimer's. By understanding the substrates and activity of ADAM10, we can gain insights into its diverse functions and contributions to disease mechanisms.

Using our improved secretome analysis, we explored the substrate specificities of ADAM10 in various cell types. Our findings reveal new dimensions of ADAM10's role in cellular processes, highlighting its potential as a therapeutic target and providing insights into the molecular mechanism of related diseases.