Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Carbohydrate-protein interactions mediate various important cellular signaling.  However, it is difficult to explore novel carbohydrate binding proteins because carbohydrate ligands often display low affinity.  Photoaffinity labeling provides a promising strategy for target identification because of its ability to capture proteins in situ by covalent bonds.  Despite the potential utility, low labeling efficiency of photoaffinity labeling and the need for the subsequent enrichment process hamper its routine use.  To address this issue, we have developed gold nanoparticle (AuNP) -based photoaffinity probes that multivalently display carbohydrate ligands and photoreactive groups at high density.  AuNPs enable rapid optimization of the probe design due to the ease of preparing the AuNP-based probes and easy protein purification by centrifugation.  It facilitated purification and dramatically improved the affinity and labeling efficiency.  Nevertheless, more efficient protein labeling is desired for target identification.  Various electrophilic groups are known as protein labeling reagent, however, there are few examples used for affinity labeling because of control of selectivity.  In this presentation, we show design and synthesis of new AuNP-based affinity labeling probes bearing lactose, a model carbohydrate ligand and various electrophilic groups for efficient capture of carbohydrate binding proteins.  We also prepared control probe presenting lactose only and evaluate its binding affinity to a lactose binding lectin PNA, to find K_d is 16 nM, which verified successful affinity enhancement by multivalent effect.  We then conducted screening of the new probes for labeling efficiency of known lectins, PNA, ECA and RCA.  Our results suggested that a judicious selection of a protein reactive group enables efficient affinity labeling of target lectins.