Spatial dynamic proteotyping of surfaceome proteostasis using DynCSC

Introduction: Protein homeostasis, known as proteostasis, ensures cellular functionality by maintaining a balanced and functional proteome. A complex network of regulatory proteostasis mechanisms maintains the proteome, which involves post-translational modifications, protein-protein interactions, and subcellular localisations. Studies have shown that spatial proteostasis in subcellular localisations cannot be elucidated based on bulk proteome studies and that spatial enrichment strategies with high specificity are needed. Spatial dynamic studies of the cell surface residing protein pool, the surfaceome, have revealed their disappearance, an essential aspect of surfaceome proteostasis. However, these studies did not include the appearance of new cell surface proteins, which is vital for fully understanding the proteostasis mechanisms of the surfaceome. Here, we developed the Dynamic Cell Surface Capturing (DynCSC) strategy, which provides system-wide insights into surfaceome proteostasis and how this information can be leveraged for regulating the surfaceome.

Method: The DynCSC strategy employs a modified triple-pulsed SILAC (Stable Isotope Labelling of Amino Acids in Cell culture) approach to capture the appearance and disappearance of cell surface receptors independently. The strategy is combined with a cell surface protein enrichment of either the global surfaceome or within a cell surface protein community using the light-mediated proximity technology LUX-MS. Using the B cell as a model system, we applied DynCSC to analyse surfaceome proteostasis using spatial proteotyping.

Results: Our findings demonstrate that surfaceome proteostasis can not be fully understood based on bulk proteome data alone. Spatially resolved cell surface proteins exhibit slower dynamics than when captured without spatial resolution. With the application of the DynCSC strategy, we further reveal that cell surface proteins reside in different temporal states and can be clustered in distinct dynamic groups. These temporal clusters are not governed by protein size, number of N-glycosylation sites and transmembrane domains. Moreover, we identify different temporal turnover kinetics within members of the B cell receptor (BCR) community, indicating independent turnover regulation within this specific protein community. Upon knocking out BCR neighbours CD81 or CD19, we observe alterations in surfaceome composition and proteostasis globally and within the BCR community locally. We specifically highlight dynamic alterations found only within the BCR community, indicating community-specific alterations.

Conclusion: DynCSC technology enables the analysis of spatially restricted proteostasis and provides new insights into the turnover of surfaceome protein communities.