Extended Data Fig. 3.

Glycoconjugation of pPY-modified proteins by CuAAC (A) Weak anion exchange (WAX) traces demonstrating successful conjugation of azHEP to form SDC2_41,55,57HEP (mint), SDC3_80,82,89HEP (pink) and SDC4_44,62,64HEP (dark purple). SDC4_44,62,64 core protein (light purple) included as reference point for SDC core proteins (. (B) Intact mass spectrometry of pPY-modified GFP (GFPyne(His)₆), used as a model protein, confirms the appropriate mass shift (+630.4 Da) when reacted with tetramethylrhodamine (TMR)-azide. (C) WAX traces after reaction of GFPyne(His)₆ with azHEP demonstrates a shift to the more anionic product (dark green). When the reaction is performed without copper (-Cu, red), the trace overlaps with WT GFP (grey). (D) WAX traces after reaction with azGAG (green) demonstrate a less anionic glycoconjugate than GFPyne + azHEP (dark green). Copper-free controls overlap with WT GFP peak. (E) SDS-PAGE analysis of WT and GFPyne(His)₆ (Y) confirms the incorporation of pPY by TMR detection only in Y. azHEP conjugation results in a higher molecular weight smear, only in the presence of copper. Representative of two technical replicates, molecular weight ladder indicates kDa. (F) Circular dichroism spectra of heparin (red), SDC1₃₇ (grey), SDC1_37HEP (orange) and HEK239T expressed mSDC1 (pink). (G) Analysis of the predicted structures from CD spectra indicates that glycosylation of SDC1₃₇ does not impart a change, or increase, in protein structures and native, glycosylated SDC1 is highly disordered.