Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry

Abstract

We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches.

Fig. 1.

PIR structure. a, conceptual modular design of novel cross-linkers, PIRs. b, the specific fragmentation pattern of PIR-labeled peptide distinguishes dead-end, intra-, and intercross-linked peptides. The neutral mass of the precursor ion equals the sum of the neutral masses of its product ions. c, structure of a biotinylated Rink-based PIR. The singly and doubly charged reporter ions are 1122.5 and 561.7, respectively. d, structure of a biotinylated DP-based PIR. The MSMS labile bonds are indicated by the dashed lines, the reactive groups are NHS esters, and the affinity group is biotin. The singly charged reporter ion is 752.4.

Fig. 2.

Diagram of two-stage mass spectrometric strategy. After in vivo labeling, protein extraction, and affinity capture of labeled proteins, enriched PIR-labeled proteins are divided into two parts. Stage 1 involves digestion and shotgun LC/MSMS for protein identification to constitute a restricted protein database. Stage 2 is performed by digesting PIR-labeled proteins first followed by another affinity enrichment of PIR-labeled peptides. Analysis of the labeled peptides is carried out with multiplexed LC/FTICR-MS. Accurate masses of the labeled peptides are measured and used for protein identification by searching against the restricted database compiled from stage 1. 2D, two-dimensional.

Fig. 3.

LC/FTICR-MS chromatograms of PIR-labeled peptides with alternating low energy (−4 V) and high energy (−22 V) applied in the collision cell. a, base peak chromatogram (BPC). b, EIC of reporter ion. The inset in a is the overlaid EICs of a PIR-labeled precursor ion (dotted line) and its released peptide ion (dashed line) and reporter ion (solid line).

Fig. 4.

An example of intercross-link determination from multiplexed spectra based on the mathematical relationship (precursor = peptide 1 + peptide 2 + reporter). a, overlaid EICs of m/z 978⁵⁺ (precursor 978⁵⁺; dotted line), m/z 1243²⁺ (peptide 1 1243²⁺; dashed bold line), m/z 1281⁺ (peptide 2 1281⁺; dashed line) and m/z 1122⁺ (reporter 1122⁺; solid line) ions, respectively. b, overlaid FTICR mass spectra extracted from scan 765 (Low Energy; solid line) and scan 766 (High Energy; dotted line). All major products resultant from cleavage of one and two labile bonds in PIR are assigned and labeled schematically.

Fig. 5.

An example of validation of intercross-link determination and peptide sequence identification for the precursor ion with neutral mass of 4884.3531. a, isolation and MS² of precursor ion (m/z 978⁵⁺). All peaks produced from cleavage of one and two PIR labile bonds are labeled schematically. b, isolation and MS³ of the released peptide ion with m/z 1243²⁺ and 1280⁺, respectively.