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. Author manuscript; available in PMC: 2024 Aug 17.
Published in final edited form as: Cell Chem Biol. 2023 Jul 17;30(8):893–905.e7. doi: 10.1016/j.chembiol.2023.06.013

Figure 1. Schematic overview of modified Glyco-SPOT synthesis.

Figure 1.

Peptide design based on the PSGL-1 sequence of human (hPSGL-1) and murine (mPSGL-1), with C-terminal and N-terminal lysines appended for bidirectional printing of peptides, and designed with N-terminal fluorescein 5(6)-FAM for downstream applications. Peptides synthesized on cellulose paper using regular Fmoc protected amino acids, Fmoc-Tyr(SO₃nP) or Fmoc-threonine with peracetylated GalNAc, core 1 or core 2. After complete synthesis, side chains were deprotected, peptide spots were cut out and peptides released by incubating with sodium bicarbonate in MeOH, which removed the neopentyls from sulfated tyrosines. Peptides were purified by HPLC and characterized on MALDI. O-glycans were elongated with a set of recombinant glycosyltransferases followed by MALDI and purified and analyzed again, to generate the final collection of structures.