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

Novo Nordisk Inflammation Research Center, Seattle, Washington, USA.
Molecular BioSystems (Impact Factor: 3.21). 06/2010; 6(6):939-47. DOI: 10.1039/b920876c
Source: PubMed


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.

Download full-text


Available from: James Bruce, Sep 01, 2015
1 Follower
26 Reads
  • Source
    • "Additionally, the labile covalent bond incorporated in the crosslinker substantially lowers the fragmentation barrier resulting in a preferential decomposition upon collision activation and safeguarding an efficient CID of high-mass precursor ions (>1500 Da). Precursor ions with elevated molecular masses, which are typically found in tryptic digests of cross-linked products, [1] [2] [3] [4] [5] [6] [7] [8] can accommodate and distribute a substantial amount of activation energy over their large number of oscillators. Hence, efficient CID fragment ion analysis is progressively hampered by an increase in molecular mass. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We have synthesized a homobifunctional active ester cross-linking reagent containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) moiety connected to a benzyl group (Bz), termed TEMPO-Bz-linker. The aim for designing this novel cross-linker was to facilitate MS analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). The TEMPO-Bz-linker was reacted with all 20 proteinogenic amino acids as well as with model peptides to gain detailed insights into its fragmentation mechanism upon collision activation. The final goal of this proof-of-principle study was to evaluate the potential of the TEMPO-Bz-linker for chemical cross-linking studies to derive 3D-structure information of proteins. Our studies were motivated by the well documented instability of the central NO―C bond of TEMPO-Bz reagents upon collision activation. The fragmentation of this specific bond was investigated in respect to charge states and amino acid composition of a large set of precursor ions resulting in the identification of two distinct fragmentation pathways. Molecular ions with highly basic residues are able to keep the charge carriers located, i.e. protons or sodium cations, and consequently decompose via a homolytic cleavage of the NO―C bond of the TEMPO-Bz-linker. This leads to the formation of complementary open-shell peptide radical cations, while precursor ions that are protonated at the TEMPO-Bz-linker itself exhibit a charge-driven formation of even-electron product ions upon collision activation. MS3 product ion experiments provided amino acid sequence information and allowed determining the cross-linking site. Our study fully characterizes the CID behavior of the TEMPO-Bz-linker and demonstrates its potential, but also its limitations for chemical cross-linking applications utilizing the special features of open-shell peptide ions on the basis of selective tandem MS analysis.
    Journal of Mass Spectrometry 02/2015; 50(2). DOI:10.1002/jms.3543 · 2.38 Impact Factor
  • Source
    • "The labile bonds can be cleaved afterwards by UV irradiation prior to the identification of interaction partners. The applications of the PIR technology have been extensively reviewed (Hoopmann, Weisbrod, and Bruce 2010; Tang and Bruce 2010; Yang et al. 2010). "
    Protein Interactions, 03/2012; , ISBN: 978-953-51-0244-1
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many essential cellular processes depend upon the self-assembly of stable multiprotein entities. The architectures of the vast majority of these protein machines remain unknown because these structures are difficult to obtain by biophysical techniques alone. However, recent progress in defining the architecture of protein complexes has resulted from integrating information from all available biochemical and biophysical sources to generate computational models. Chemical cross-linking is a technique that holds exceptional promise toward achieving this goal by providing distance constraints that reflect the topography of protein complexes. Combined with the available structural data, these constraints can yield three-dimensional models of higher order molecular machines. However, thus far the utility of cross-linking has been thwarted by insufficient yields of cross-linked products and tandem mass spectrometry methods that are unable to unambiguously establish the identity of the covalently labeled peptides and their sites of modification. We report the cross-linking of amino moieties by 1,3-diformyl-5-ethynylbenzene (DEB) with analysis by high resolution electron transfer dissociation. This new reagent coupled with this new energy deposition technique addresses these obstacles by generating cross-linked peptides containing two additional sites of protonation relative to conventional cross-linking reagents. In addition to excellent coverage of sequence ions by electron transfer dissociation, DEB cross-linking produces gas-phase precursor ions in the 4+, 5+, or 6+ charge states that are readily segregated from unmodified and dead-end modified peptides using charge-dependent precursor selection of only quadruply and higher charge state ions. Furthermore, electron transfer induces dissociation of the DEB-peptide bonds to yield diagnostic ion signals that reveal the "molecular ions" of the unmodified peptides. We demonstrate the power of this strategy by cross-linking analysis of the 21-protein, ADP-bound GroEL-GroES chaperonin complex. Twenty-five unique sites of cross-linking were determined.
    Molecular &amp Cellular Proteomics 10/2010; 9(10):2306-17. DOI:10.1074/mcp.M110.003764 · 6.56 Impact Factor
Show more