Interaction Partners to Short Modified Motifs

One of the main interests in signaling biology is to gain insight into how cells process information. In many signaling pathways, signaling molecules bind to receptor proteins and this information is passed on to ultimately induce tran-scriptional changes, often involving a series of protein phosphorylation events. A significant subset of these signal-dependent phosphorylation events creates new protein-protein interaction interfaces, which serve to recruit adaptor or effector molecules. Mechanistically, signal transduction pathways in all organisms are composed of modification-dependent protein-protein interactions between domains and extended peptide motifs. Consensus peptide sequences recognized by different protein domains have been studied in the past using oriented peptide libraries, peptide arrays, or phage display (Elia et al. 2003; Espejo et al. 2002; Kay et al. 2001). However, most of these methods lack specificity for modification-dependent interactions and few of them are unbiased or can be performed without already having candidate target proteins.

The use of synthetic modified and unmodified peptides as a bait in pulldown experiments to find peptide motif-based interaction partners appears to be a powerful method to dissect the role of certain peptide motifs in signaling events (Fig. 1). In this approach, synthetic peptide pairs comprising a motif of interest in modified (e.g., phosphorylated) and unmodified (e.g., unphosphorylated) form are used as bait in pull-down experiments. Eluted proteins are identified by mass spectrometry, and specific interaction partners are defined as being identified exclusively in pull-downs with the modified bait peptide and not the unmodified control bait peptide. This al-

Fig. 1 Principle of modification-dependent interaction screening. Two protein populations are obtained by metabolic labeling (Engelsberger et al. 2006) and subjected to pull-down experiment using modified (indicated by P) and unmodified synthetic peptides as a bait. The bait peptides are chosen around a specific site of phosphorylation in a protein of interest. After incubation of protein extracts with the bait peptide of interest, eluted proteins are combined and digested with trypsin. Tryptic peptides are then detected by mass spectrometry. Those tryptic peptides from proteins specifically binding the phosphory-lated bait will have a larger peak intensity of the 15N-labeled form. Nonspecific binders will have a 1: 1 ratio of both isotopic forms

Fig. 1 Principle of modification-dependent interaction screening. Two protein populations are obtained by metabolic labeling (Engelsberger et al. 2006) and subjected to pull-down experiment using modified (indicated by P) and unmodified synthetic peptides as a bait. The bait peptides are chosen around a specific site of phosphorylation in a protein of interest. After incubation of protein extracts with the bait peptide of interest, eluted proteins are combined and digested with trypsin. Tryptic peptides are then detected by mass spectrometry. Those tryptic peptides from proteins specifically binding the phosphory-lated bait will have a larger peak intensity of the 15N-labeled form. Nonspecific binders will have a 1: 1 ratio of both isotopic forms lows identification of specific binding partners to the modified bait peptides, even in the presence of a large excess of background binders. Background proteins are characterized by also being present in the pull-down with the control peptide (Schulze and Mann 2004).

This peptide-protein interaction screen is specific and reproducible, as shown by a medium-scale analysis of the complete phosphotyrosine interac-tome of the ErbB-receptor tyrosine kinase family (Schulze et al. 2005), and in a proof-of-principle study using protein extracts from plant cell cultures, a 14-3-3 protein was identified as interaction partner to a bait peptide comprising the 14-3-3 binding site of nitrate reductase (Gruhler et al. 2005).

Compared with two-hybrid and array-based approaches, this peptide pulldown strategy has the advantages that fully processed and modified sequences can serve as baits, and that the interaction takes place in the native environment of the cell extract. However, it has to be kept in mind that mass spectrometry based affinity methods will only be able to detect a subset of the protein interactions that actually occurs in vivo.

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