A new mass spectrometry identifiable cross-linking strategy has been developed to study protein-protein interactions. The new cross-linker was designed to have two low-energy MS/MS-cleavable bonds in the spacer chain to provide three primary benefits: First, a reporter tag can be released from cross-link due to cleavage of the two labile bonds in the spacer chain. Second, a relatively simple MS/MS spectrum can be generated owing to favorable cleavage of labile bonds. And finally, the cross-linked peptide chains are dissociated from each other, and each then can be fragmented separately to get sequence information. Therefore, this novel type of cross-linker was named protein interaction reporter (PIR). To this end, two RINK groups were utilized to make our first-generation cross-linker using solid-phase peptide synthesis chemistry. The RINK group contains a bond more labile than peptide bonds during low-energy activation. The new cross-linker was applied to cross-link ribonuclease S (RNase S), a noncovalent complex of S-peptide and S-protein. The results demonstrated that the new cross-linker effectively reacted with RNase S to generate various types of cross-linked products. More importantly, the cross-linked peptides successfully released reporter ions during selective MS/MS conditions, and the dissociated peptide chains remained intact during MS(2), thus enabling MS(3) to be performed subsequently. In addition, dead-end, intra-, and inter-cross-linked peptides can be distinguished by analyzing MS/MS spectra.

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 ( 1 or a single (e.g. FLAG tag (2)) or double affinity tag (e.g. TAP tag (3, 4)) followed by protein identification with mass spectrometry, protein microarray technology (5, 6), and computational prediction methods (7,8). Although all these approaches demonstrate great promise in mapping protein-protein interactions on a proteome wide level, the resulting large scale data sets are often associated with high rates of false negatives and false positives (Ͼ50%), and poor overlap of data sets among different approaches used for the same system are often observed (9 -11). Such observations suggest that no single method is flawless and comprehensive. The strengths and weaknesses of each method have been thoroughly reviewed (12-15). For example, traditional IP-based affinity purification methods require a specific antibody for every protein of interest that is a hindrance for widespread, large scale application. Tag-based methods overcome this limitation by fusing the bait protein genetically with an affinity tag that is applicable to all proteins. One of the most successful tagbased methods is TAP technology, which fuses two affinity tags to the bait protein, and nonspecific binding is significantly reduced with two sequential purification steps (3, 4). Although tag-based methods allow bait proteins to be expressed in vivo and interact with native physiological partners, recent studies showed that tagging can also cause overexpression of the bait protein that can result in association with chaperones and improper intercellular localization (16,17). In addition, tagging one bait protein at a time for large scale studies can be tedious and costly. Another issue worth noting is that all affinity-based methods require cell lysis prior to purification of the associated complex of the bait protein.During cell lysis, the native cellular system is disturbed, and the bait protein is present in the lysis buffer, which is very different from the intracellular milieu. As described recently by Berggard et al. (13), the fact that the affinity between interacting proteins may be substantially different in vivo as comFrom the ‡Department

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.

Protein interactions and topologies are key features that enable specificity, function and the evolution of highly integrated, regulated networks in biological systems. Primary challenges associated with the study of biological systems include identification of protein interactions and measurement of topological features of proteins and their interactions in vivo. Advancements such as the Yeast Two-Hybrid (1), coimmunoprecipitation (2), and Tandem Affinity Purification tags (3) have greatly increased the ability to identify hundreds or even thousands of interactions from complex biological samples (2, 4 -6). Despite the many thousands of protein interactions that are now known (7) however, for only a tiny fraction is there any knowledge of their in vivo topology. On the other hand, if topologies of interactions were more widely known, this information could improve understanding of underlying fundamental factors that drive interactions, improve development of highly specific modulators of protein interactions, improve interaction prediction capabilities, and improve comprehension on biological systems. Unfortunately, exceedingly few methods exist to allow unbiased measurement of proteinprotein interaction topological features in cells.Chemical cross-linking has great potential for in vivo interaction topological studies (8 -10). Cross-linked peptides contain information about interacting protein identities and can uniquely define regions of protein sequences that are near one another when proteins are present within the native cellular environment. Challenges associated with in vivo crosslinking analysis that have precluded this achievement include the difficulty in identification of cross-linked peptides and the severe dynamic range constraints resultant from the overwhelming majority of noncross-linked peptides. Our efforts to overcome these challenges resulted in development of Protein Interaction Reporter (PIR) 1 technology (11) that uses a novel type of cross-linker and mass spectrometry to identify peptides that are close to one another within protein complexes in cells. These efforts resulted in the first reported identification of cross-linked peptides from live cells (9) including the first in vivo identification of an interaction among two outer membrane cytochrome c proteins, an interaction that appears to be critical to electron transport properties of Shewanella oneidensis (12).Here we present the first application of PIR technology to the study of interactions in E. coli cells where 65 cross-linked peptide pairs were unambiguously identified. To date, this constitutes the largest in vivo cross-linked peptide data set ever produced. In this system, we are also able to compare many of our results with known protein and protein complex crystal structures that demonstrate excellent agreement with our in vivo data. Importantly, this comparative analysis was also used to define distance constraints that enable refinements of structural prediction of in vivo protein complexes never before possible. Furthe...

Triggering receptor expressed on myeloid cells (TREM)-1 is an orphan receptor implicated in innate immune activation. Inhibition of TREM-1 reduces sepsis in mouse models, suggesting a role for it in immune responses triggered by bacteria. However, the absence of an identified ligand has hampered a full understanding of TREM-1 function. We identified complexes between peptidoglycan recognition protein 1 (PGLYRP1) and bacterially derived peptidoglycan that constitute a potent ligand capable of binding TREM-1 and inducing known TREM-1 functions. Interestingly, multimerization of PGLYRP1 bypassed the need for peptidoglycan in TREM-1 activation, demonstrating that the PGLYRP1/TREM-1 axis can be activated in the absence of bacterial products. The role for PGLYRP1 as a TREM-1 activator provides a new mechanism by which bacteria can trigger myeloid cells, linking two known, but previously unrelated, pathways in innate immunity.