Protocol to quantify site-specific carbohydrate sulfation at immune checkpoint proteins in mammalian cells

Carbohydrate sulfotransferase 8 (CHST8) promotes immune checkpoint activation by sulfating GalNAcβ1-4GlcNAc (LacdiNAc) termini on glycoproteins. We present a protocol for quantifying LacdiNAc levels in mammalian cells using a lectin-captured luciferase reporter assay. We describe steps for preparing plasmid encoding NanoLuc-fused protein and lectin-coated multiple well plates, expressing NanoLuc-fused protein, and detecting luciferase activity. We then detail procedures for the lectin-capture assay and analyzing data after luminescence signals are measured. This protocol is adaptable for other glycoforms by substituting appropriate lectins. For complete details on the use and execution of this protocol, please refer to Chou et al.¹.