Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Microbial Genomics
88
A Protein Interaction Reporter Strategy for Systems-Level Protein Interaction Networks of Shewanella oneidensis MR-1
James E. Bruce1* (james_bruce@wsu.edu), Xiaoting Tang1, Harry Zhu1, Saiful Chowdhury1, Devi Adhikari1, Gerhard Munske1, Gordon A. Anderson2, and Nikola Tolic2
1Washington State University, Pullman, WA and 2Pacific Northwest National Laboratory, Richland, WA
A key challenge inherent in the utilization of genomic data is related to deciphering the network of protein-protein interactions that enable biological function. Chemical cross-linking has gained increasing interest as a tool for protein interaction profiling, yet the examples of successful application are relatively scarce. The difficulty inherent in mass spectral interpretation and protein identification resultant from cross-linking reaction mixtures is a significant barrier that has hindered many such efforts. The analysis of complex MS and MS/MS patterns resultant from various types of cross-linker products and multiple fragmentation pathways can present levels of complexity that preclude protein and protein-protein interaction identification. However, a general technique that can identify proteins based on a physical property common to protein-protein interactions, namely, the proximity of multiple protein species within a complex mixture, is still very desirable.
Our efforts have been devoted to the development of a novel approach for chemical cross-linking that can enable improved identification of protein interactions in complex systems. A key component of this research is the development of new compounds that can provide advanced features and additional information from cross-linking reaction mixtures. We call our approach that employs mass spectrometry-cleavable cross-linkers a “Protein Interaction Reporter” (PIR) strategy, since the fragments of the cross-linker themselves are encoded with additional information that enables improved analytical capabilities for protein interaction profiling. For example, our first-generation PIR structures have been developed with low-energy CAD cleavable bonds that, when activated, release a reporter ion of specific m/z. These bonds can be efficiently fragmented at energy levels that preclude fragmentation of nearly all peptide amide backbone bonds. Thus, our initial MS/MS analyses of PIR-labeled products are less congested by complex multiple fragmentation pathways that are commonly observed in most cross-linked peptide MS/MS spectra. Next-generation PIR structures employ additional fragmentation schemes and features to allow even more information to be encoded in the compound. In all cases, the measured m/z’s of fragment ions resultant from PIR-peptide complex activation provide information that enables improved cross-link type and cross-linked peptide identification.
This presentation will highlight our initial proof-of-principle PIR experiments that were performed with model noncovalent complexes. These applications of PIR technology showed that MS/MS data could be used to differentiate various product types from cross-linking reactions, and help pinpoint ions that are resultant from protein interactions. Since the complexity of products normally poses a significant impediment to successful cross-linker application even for model noncovalent complexes, PIR advancements allow improved capabilities for analysis of protein interactions with mass spectrometry. This approach was able to produce protein-protein interaction structural data in excellent agreement with the known X-Ray crystal structure of Ribonuclease S, our model noncovalent complex. In addition, second-generation PIR structures were synthesized to incorporate affinity capture capabilities to allow enrichment of cross-linking products from complex mixtures. This feature will allow PIR cross-linked products to be enriched from digestion mixtures of proteins. Our initial investigations with this compound demonstrated that the sites of incorporation of a biotinylated PIR were nearly identical to those of the first generation structure. Thus, the affinity label seems to pose no major limitation to the reactivity of the PIR. We have also begun application of this PIR structure to complex protein mixtures, including Shewanella oneidensis MR-1 cell lysates in an effort to better define conditions for cross-linking studies with our compounds. The results of these initial applications, additional compound features, and our envisioned implementation of the PIR strategy with tandem accurate mass analyses for the characterization of protein interactions in Shewanella oneidensis MR-1 will be presented.
This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-04ER63924.
* Presenting author
