Residue-specific N-terminal glycine to aldehyde transformation renders analytically pure single-site labeled proteins.

Here, we present N-Gly-specific glyoxamide generation in native proteins, isolated or in a complex mixture. The resulting aldehyde enables parallel installation of probes and a purification platform to render analytically pure single-site tagged proteins. It renders N-Gly engineered insulin without perturbing its structure, receptor binding, and downstream signaling pathway.