Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars.
Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4-6 d, and photocrosslinking experiments can be completed in an additional 6 d.
Bond
Michelle R
MR
Department of Chemistry, Stanford University, Stanford, California, USA.
Zhang
Haochi
H
Vu
Peter D
PD
Kohler
Jennifer J
JJ
D018929:Cell Culture Techniques; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003978:Diazomethane / Q000737:chemistry*; D005434:Flow Cytometry; D006001:Glycoconjugates / Q000737:chemistry* / Q000302:isolation & purification*; D015394:Molecular Structure; D010777:Photochemistry / Q000379:methods*; D012794:Sialic Acids / Q000737:chemistry; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D006001:Glycoconjugates; D012794:Sialic Acids; D003978:Diazomethane