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.

Figure 1

(Left) A flowchart that describes how the REACT algorithm functions during LC–MSⁿ experiments. (Right) An idealized practical diagram of how the algorithm would operate on real data directly corresponding to the flowchart.

Figure 2

An example of ReACT data acquired from PIR labeled E. coli cells. (A) High resolution MS acquisition for precursor information; inset is an expanded view of the spectrum surrounding the cross-linked peptide precursor, 718.174 m/z. (B) High resolution MS² acquisition for cross-linked peptide relationship information. (C and D) Low resolution MS³ acquisition for peptide sequence information. (E) Tryptophanase crystal structure (E. coli, PDB 2OQX) with all observed cross-linked sites marked in gray; the cross-link observed in this data is marked in red, while other sites observed in additional cross-linked sites are in gray. To view an animated illustration of cross-linked sites on the molecular structure see: http://brucelab.gs.washington.edu/ReACT_movies.php.

Figure 3

(A) High resolution MS² spectra acquired on a cross-linked species with two different cross-linkers within the same LC-ReACT experiment. The cross-linked site identified involves the same two peptides from RNase A (ETAAAKFER and NLTKDR). The top contains this site identified with BDP cross-linker (blue), and the bottom contains this site identified with 2Rink cross-linker (red). Low resolution MS³ used to make peptide sequence identification for NLTKDR (B) and ETAAAKFER (C) for both linkers.

Figure 4

(A) Interaction network comprising cross-linked information obtained using ReACT on E. coli cells. Node colors represent the subcellular localization for the proteins identified in cross-linked sites. Labeled nodes represent “hubs” for which many cross-links between proteins were detected. (B) Subcellular localization of cross-linked proteins.

Figure 5

E. coli 30s ribosome (PDB 3FIH) with 3 of 4 observed interprotein ribosomal cross-links mapped (RNA has been omitted). To view an animated illustration of cross-linked sites on the molecular structure see: http://brucelab.gs.washington.edu/ReACT_movies.php.