Chemical crosslinking enables profiling of endogenous cell surface galectin-3 interactions

Galectin-3 is a {beta}-galactoside-binding lectin that organizes glycoproteins into cell surface lattices (multivalent interaction networks) that regulate biology spanning immune function, fibrosis, heart disease, and cancer progression. Galectin-3 engages promiscuous but low-affinity glycan ligands, posing technical challenges for faithfully mapping the cell surface galectin-3 interactome. Here, we introduce Crosslinking-assisted Galectin Lattice Affinity Purification Mass Spectrometry (xGL-APMS), a facile and stringent proteome-scale approach that captures galectin-3 interactions natively at the cell surface, without the need for exogenous galectin-3, and is extendable to XL-MS. We identified 125 N-glycan branching-dependent surface galectin-3 interactors on Jurkat cells, validated interactions with six orthogonal approaches, and compared low and high-dose galectin-3 interactomes, revealing differences in both distribution and interactome composition. Finally, without exogenous or modified bait, we performed immediate crosslinking of endogenous galectin-3 to identify 69 galectin-3-associated proteins on primary mouse thymocytes. Together, these approaches provide a readily adoptable framework for measuring endogenous surface galectin-3 and profiling its interacting partners.