Comparative analysis of photocatalytic, enzymatic, and chemical approaches to identify canonical and noncanonical surfaceomes.

The cell surface is a critical nexus in cellular communication and a rich landscape yielding the majority of current therapeutic targets, yet only a small fraction of cell surface proteins have been targeted therapeutically. The cell surface proteome (surfaceome) comprises only ~1% of the human proteome by mass, motivating enrichment strategies to detect surfaceome alterations in disease for drug target discovery. Developments in photocatalytic technology have revolutionized the study of protein neighborhoods around specific targets of interest, but photocatalysis has been underutilized for global surfaceome analysis. Here, we describe five distinct photocatalytic surfaceomics strategies, all without the need for cellular engineering, that apply the photocatalyst Eosin Y attached to different cell surface structures: direct glycan ligation, glycan attachment via two different lectins, membrane insertion, and free in extracellular space. We systematically compare these photocatalytic surfaceomics methods to each other as well as to three established chemical and enzymatic surfaceomics methods. We demonstrate that the different photocatalytic surfaceomics techniques achieve broadly similar canonical cell surface protein enrichment but show some important differences reflective of cell surface attachment mechanism. These mechanistically orthogonal surfaceomics techniques consistently identified numerous nonglycosylated proteins with high concordance, many of which represent an underappreciated class of noncanonical cell surface proteins lacking traditional cell surface protein features that may represent potential drug targets. This study substantially expands options for surfaceomics methods using photocatalytic proximity labeling.