Abstract
Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs). In many cases, these PPIs are between a folded protein domain and a short linear motif (SLiM) within an unstructured region of a protein. As a result of this small interaction interface (3-10 amino acids), the affinities of SLiM-mediated interactions are typically weak (K ds of ~1-10 µM), allowing physiologically relevant changes in cellular concentrations of either protein partner to dictate changes in occupancy and thereby transmit cellular signals. However, these weak affinities also render detection and quantitative measurement of these interactions challenging and labor intensive. To address this, we recently developed MRBLE-pep, a technology that employs peptide libraries synthesized on spectrally encoded hydrogel beads to allow multiplexed affinity measurements between a protein and many different peptides in parallel. This approach dramatically reduces both the amount of protein and peptide as well as the time required to measure protein-peptide affinities compared to traditional methods. Here, we provide a detailed protocol describing how to: (1) functionalize polyethylene glycol diacrylate (PEG-DA) MRBLE beads with free amine groups, (2) synthesize peptide libraries on functionalized MRBLEs, (3) validate synthesized peptide sequences via MALDI mass spectrometry and quantify evenness of peptide coverage on MRBLEs, (4) use MRBLE-bound peptide libraries in multiplexed protein binding assays, and (5) analyze binding data to determine binding affinities. We anticipate that this protocol should prove useful for other researchers seeking to use MRBLE-pep in their own laboratories as well as for researchers broadly interested in solid-phase peptide synthesis and protein-protein binding assay development.
Highlights
[Background] Cellular signal transduction pathways rely on protein-protein interactions (PPIs)
The majority of these interactions are mediated by short linear motifs (SLiM) in disordered regions of proteins and a globular domain on another protein (Neduva and Russell, 2006; Dinkel et al, 2016)
The polyethylene glycol diacrylate (PEG-DA) polymers that make up MRBLEs hydrogel beads must be chemically functionalized with free amine groups to allow direct coupling of Fmoc-protected amino acid building blocks
Summary
This complete protocol describes (Figure 1): (1) Functionalization of PEG-DA hydrogel beads in preparation for peptide synthesis, (2) solid-phase Fmoc peptide synthesis on PEG-DA beads, (3) peptide validation and peptide density measurements, (4) multiplexed protein-peptide binding assays using bead-bound peptide libraries, and (5) final data analysis to extract quantitative binding affinity information (Kd). In this protocol, all amounts are calculated for 48 tubes containing one code each (a 48-code peptide library) with an estimated 1 mg of MRBLEs for each code (about 10,000 beads) and a loading capacity of 0.32 mmol/g.
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