In Vivo Protein Interaction Network Identified with a Novel Real-Time Cross-Linked Peptide Identification Strategy

Abstract

Protein interaction topologies are critical determinants of biological function. Large-scale or proteome-wide measurements of protein interaction topologies in cells currently pose an unmet challenge that could dramatically improve understanding of complex biological systems. A primary impediment includes direct protein topology and interaction measurements from living systems since interactions that lack biological significance may be introduced during cell lysis. Furthermore, many biologically relevant protein interactions will likely not survive the lysis/sample preparation and may only be measured with in vivo methods. As a step toward meeting this challenge, a new mass spectrometry method called Real-time Analysis for Cross-linked peptide Technology (ReACT) has been developed that enables assignment of cross-linked peptides “on-the-fly”. Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked E. coli cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application. Of the identified interprotein cross-linked peptide pairs, 40% are derived from known interactions and provide new topological data that can help visualize how these interactions exist in cells. Other identified cross-linked peptide pairs are from proteins known to be involved within the same complex, but yield newly discovered direct physical interactors. ReACT enables the first view of these interactions inside cells, and the results acquired with this method suggest cross-linking can play a major role in future efforts to map the interactome in cells.

Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions

“…This can be achieved by the comparison of systematic interaction changes upon a specific stimuli or perturbation using cross-linking snapshots. Some pioneer studies have been applied in bacterial cells [8,10,24]. Another direction is quantitative crosslinking.…”

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

“…Protein interaction reporter (PIR) technology has emerged as a powerful approach to probe in vivo and in vitro interactions between and within multimeric protein complexes (16,18,19). The strategy was developed to identify amino acid residues in interacting proteins that exist close to one another (typically, Յ35Å) by coupling chemical cross-linking with high-resolution mass spectrometry (MS) to derive peptide sequence information.…”

“…Sequence identification of affinity captured cross-linked peptide pairs is achieved using Fourier transform ion cyclotron resonance (FT-ICR) MS coupled with Real-time Analysis Cross-link Technology (ReACT). The MS-labile bonds in PIR molecules allow for the controlled release of the cross-linked peptides within the mass spectrometer so that fragmentation of the peptide backbone can occur on each peptide individually (16,(18)(19)(20). The ReACT algorithm allows for the tandem identification of two cross-linked peptides by validating the PIR mass relationship (precursor ϭ mass peptide 1 ϩ mass peptide 2 ϩ mass reporter) of the original cross-linked species in real time.…”

“…ship are selected for peptide fragmentation (19). The resulting spectra are mined for protein sequence information using traditional proteomic search engines such as Mascot (21) and SE-QUEST (22).…”

“…The resulting spectra are mined for protein sequence information using traditional proteomic search engines such as Mascot (21) and SE-QUEST (22). PIR-derived cross-linked sites identified between or within proteins are then used as distance constraints to develop structural models that enable visualization of proteins and protein complexes, as they exist in cells during cross-linker application (19,23,24). A recent study using in vivo PIR application confirmed OmpA homodimer existence and interfacial regions that were originally identified in bacterial cells by traditional MS methods (25), demonstrating the ability to derive meaningful structural information from this experimental approach.…”