Peptide array–based interactomics

Abstract

The analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based techniques, peptide pull-downs are an alternative tool specifically useful to study interactome changes induced by post-translational modifications. Peptides for pull-downs can be chemically synthesized and thus offer the possibility to include amino acid exchanges and post-translational modifications (PTMs) in the pull-down reaction. The combination of peptide pull-down and analysis of the binding partners with mass spectrometry offers the direct measurement of interactome changes induced by PTMs or by amino acid exchanges in the interaction site. The possibility of large-scale peptide synthesis on a membrane surface opened the possibility to systematically analyze interactome changes for mutations of many proteins at the same time. Short linear motifs (SLiMs) are amino acid patterns that can mediate protein binding. A significant number of SLiMs are located in regions of proteins, which are lacking a secondary structure, making the interaction motifs readily available for binding reactions. Peptides are particularly well suited to study protein interactions, which are based on SLiM-mediated binding. New technologies using arrayed peptides for interaction studies are able to identify SLIM-based interaction and identify the interaction motifs. Graphical abstract

Using PrISMa to reveal the interactome of the human claudins family

“…Hence, all the interactors must be confirmed using other methods. 6 In previous studies, proximity ligations, co-immunoprecipitations, and Bio-ID assays successfully confirmed several interaction partners discovered by PrISMa. 3 , 5 …”

“… 1 , 2 , 3 , 4 , 5 This protocol describes the PrISMa approach as a screening method to target interactions mediated by short linear motifs (SLiMs) in high-throughput. 5 , 6 Since the peptide matrix is chemically synthesized, it allows the inclusion of modified amino acids, like amino acids carrying post-translational modifications or the inclusion of amino acid exchanges caused by mutations.…”

“…However, this can be applied to any protein or region of interest with high content in intrinsically disordered structure. 6 Peptides should be around 13–15 amino acids long and tiled by 3–5 amino acids to the previous peptide ( Figure 1 ). PTMs and/or mutations are included as separate entries.…”

“…PTMs and/or mutations are included as separate entries. 6 After designing the screen matrix, the chosen peptides are synthesized by Fmoc (Fluorenylmetyloxicarbonyl protecting group) chemistry in solid-phase peptide synthesis (SPOT) 9 , 10 onto a PEG-cellulose membrane. 11 In the second step, we isolate the protein lysate from MDCK-C7 cells needed for the PrISMa pull-down.…”

Epitopes and Mimotopes Identification Using Phage Display for Vaccine Development against Infectious Pathogens

“…Peptide arrays typically consist of a series of short peptides, each representing a potential epitope of the antigen of interest, and helps to recognize binding sites between proteins [ 12 , 13 , 14 , 15 ]. By incubating the peptide array with sera or cells from an individual with a known immune response to the antigen, it is possible to identify the specific peptides that are recognized by the immune system.…”