SPOT peptide synthesis
Generation of peptides for structure–activity relationship studies is generally achieved in a robust and efficient manner by applying standard Fmoc/tBu solid-phase peptide synthesis (SPPS). However, for screening of larger numbers of peptides (>20–30) toward a given target, standard bead-based SPPS becomes expensive and time-consuming, due to the use of reagents and the requirement for preparative HPLC purification. Therefore, technologies aiming to miniaturize and simplify this process are desirable and one such example is SPOT peptide synthesis.
Picture: (Alt text for picture: https://go.nature.com/3nHe7sT)
In SPOT peptide synthesis, peptides are synthesized using the Fmoc/tBu strategy with their C-termini covalently attached to a cellulose membrane, which can be directly applied in a target binding assay. Alternatively, modified cellulose disks can be used that are partially degraded in acid, creating a soluble peptide-cellulose conjugate that can be printed onto coated glass slides (Figure above). With µSPOT, the peptide-cellulose stock solutions can be printed to generate hundreds of microarray slides ready for testing.
At the Center for Biopharmaceuticals, we routinely apply µSPOT methodology and have recently published several examples of its application for mapping peptide–protein interactions to discover high-affinity peptide-based binders. For example, discovery of gephyrin-binding peptides to visualize postsynaptic sites and modulate neurotransmission (Strømgaard group).1 Also, peptide based-inhibitors targeting the APP–Mint2 protein–protein interaction to reduce amyloid-β formation has been discovered (Strømgaard group).2 Furthermore, in an effort to map the interactions of histone deacetylase (HDAC) enzymes with their histone substrates, including the effect of posttranslational modifications, we identified peptide binders with activity in cells (Olsen group).3
Through the µSPOT technology, the Center for Biopharmaceuticals performs high-throughput synthesis and testing of peptide libraries to develop new research tools and drug candidates.
- Maric, H.M., Hausrat, T.J., Neubert, F., et al. Gephyrin-binding peptides visualize post-synaptic sites and modulate neurotransmission. Nat. Chem. Biol. 13, 153–160 (2017).
- Bartling, C.R.O., Jensen, T.M.T., Henry, S.M., Colliander, A.L., et al. Targeting the APP-Mint2 protein–protein interaction with a peptide-based inhibitor reduces amyloid-β formation. J. Am. Chem. Soc. 143, 891–901 (2021).
3. Moreno-Yruela, C., Bæk, M., Vrsanova, A.-E. et al. Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases. Nat Commun 12, 62 (2021).