The application of modular protein domains in proteomics

Abstract

The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as "domainomics" to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction.

Fig. 1

Domainomics assay designs. The three basic assay designs for studying interactions between modular protein domains and short peptide motifs are depicted. Top: In motif scanning, a domain of interest is used to probe a library of peptide motifs or proteins containing binding motifs, typically to define domain specificity or identify possible binding proteins. For example, an immobilized domain can be used as bait in pull-downs. Middle: In domain scanning, a motif of interest is used as a probe to screen a set of domains or domain-containing proteins. Bottom: Multiplex scanning simultaneously assesses interactions between many ligands and domains, providing the specificities of domains within a domain-motif interaction map. Multiplex scanning can be designed as an expanded version of domain or motif scanning, or as a “library to library pull-down” to screen for binding modules (see Section 5.3.).

Fig. 2

Domainomics technologies. (A) Forward phase array. Modular domains are immobilized on a solid support and probed with a peptide motif or protein (domain scanning). Duplicate arrays can be probed with different peptides (multiplex scanning). Binding is detected through a peptide-conjugated tag using fluorescence- or enzyme-based systems. (B) Reverse phase array. Peptides or proteins are immobilized on a solid support and probed with a tagged domain (motif scanning). Duplicate arrays can be probed with different domains (multiplex scanning). Binding is detected through a domain-conjugated tag using fluorescence- or enzyme-based systems. (C) Phage display. Peptide libraries are expressed as bacteriophage coat protein fusions and incubated with an immobilized domain (motif scanning). High affinity ligands are identified by sequencing of phage DNA. Multiple domains can be studied in separate wells with replicate libraries (multiplex scanning). Domains expressed on the phage surface can also be incubated with an immobilized peptide or protein (domain scanning). (D) Bead array. A bead-bound peptide library is probed with a tagged domain (motif scanning). Duplicate arrays can be probed with different domains (multiplex scanning). Binding is detected through a conjugated tag using fluorescence-, enzyme-, or mechanical-based systems. Peptide sequences are identified via mass spectrometry. Domains can also be immobilized on beads and probed with a motif (domain scanning). (E) In vivo photocrosslinking. This approach allows for incorporation of a photocrosslinking amino acid, e.g., p-benzoyl-L-phenylalanine (pBpa) into a modular domain expressed in vivo. Bound ligands are crosslinked to the domain under UV light, subjected to a pull-down assay using an affinity tag, and identified by MS. Covalent binding between the domain and ligands improves identification of weak transient binders, and allows for removal of indirect binding proteins by rigorous washing steps.