Mapping the binding sites on viral proteins that interact with host glycans is essential for understanding host–pathogen interactions and guiding therapeutic design. While the SARS-CoV-2 spike (S) protein is known to bind host glycans through its N-terminal domain and receptor-binding domain (RBD), the specific glycan-interacting residues on the RBD remain undefined. Here, we introduce antibody masking-native mass spectrometry (AM-nMS), a spatially resolved assay for mapping glycan-binding sites by combining a panel of nanobodies targeting distinct regions within the RBD with center-of-mass monitoring and catch-and-release strategies. Applied to wild-type (Wuhan-Hu-1) SARS-CoV-2 RBD, AM-nMS reveals distinct yet partially overlapping glycan-binding surfaces. Heparan sulfate (HS) oligosaccharides bind within a shallow groove (encompassing residues 420–430, 441–452, and 490–512), which partially overlaps the receptor-binding motif (RBM). Sialylated glycans, including ganglioside oligosaccharides, engage a distal edge of the RBM (residues 470–486 and 490–512) that includes a portion of HS binding groove. Globoside glycans bind at a third site (residues 513–530 and 441–452) that also partially overlaps with the HS-binding region. These multiple binding sites near the RBM enable the RBD to interact with a wide range of host glycans. Site assignments are supported by library screening across the RBD from several SARS-CoV-2 variants of concern (VOCs), competitive binding nMS, mass photometry and molecular dynamics simulations. This work presents the first experimental residue-level map of glycan engagement by the SARS-CoV-2 RBD and establishes AM-nMS as a broadly applicable strategy for localizing glycan-binding sites on proteins.

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