A new cross-linking strategy: protein interaction reporter (PIR) technology for protein-protein interaction studies

Abstract

Chemical cross-linking coupled with mass spectrometry, an emerging approach for protein topology and interaction studies, has gained increasing interest in the past few years. A number of recent proof-of-principle studies on model proteins or protein complex systems with improved cross-linking strategies have shown great promise. However, the heterogeneity and low abundance of the cross-linked products as well as data complexity continue to pose enormous challenges for large-scale application of cross-linking approaches. A novel mass spectrometry-cleavable cross-linking strategy embodied in Protein Interaction Reporter (PIR) technology, first reported in 2005, was recently successfully applied for in vivo identification of protein-protein interactions as well as actual regions of the interacting proteins that share close proximity while present within cells. PIR technology holds great promise for achieving the ultimate goal of mapping protein interaction network at systems level using chemical cross-linking. In this review, we will briefly describe the recent progress in the field of chemical cross-linking development with an emphasis on the PIR concepts, its applications and future directions.

Fig. 1

Cross-linking applications: targeted interactions and large-scale interactions.

Fig. 2

PIR structure. (a) Conceptual modular design of novel cross-linkers, protein interaction reporters (PIRs). The functional groups are color coded with structure examples given in (c) and (d). (b) The specific fragmentation pattern of PIR-labeled peptide distinguishes dead-end, intra-, and inter-cross-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 and (d) structure of a biotinylated DP-based PIR. The MS/MS labile bonds are indicated by the red dashed lines, the reactive groups are NHS-esters highlighted in blue and the affinity group is biotin highlighted in yellow.

Fig. 3

An example of inter-cross-link determination from multiplexed FTICR-MS spectra based on the mathematical relationship (precursor = peptide 1 + peptide 2 + reporter). A biotinylated Rink-based PIR (Fig. 2c) is used. The given example is a cross-linked peptide pair from protein MtrC and SO0404 in Shewanella oneidensis.

Fig. 4

An example of MSMS spectrum of the peptide ion released from CID activation of cross-linked precursor ion using an ion trap mass spectrometer. The remaining stump modification on residue K is C4H5N1O2 with a mass of 99.089.

Fig. 5

Incorporation of stable isotopes in PIR structure will result in heavy and light versions that can be used for relative quantitation among multiple samples. * indicates ¹³C and/or deuterium-substituted compound available as a building block from commercial sources. The resultant PIR pairs will differ by 6 to 8 Daltons, depending on which site turns out to be optimal for isotope incorporation. The mass differentiation of labeled peptides from each sample will allow relative quantitation based on observed peak intensities.