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 ( 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

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

“…Using the protein interaction reporter concept based on gas phase cleavable, affinity tagged crosslinking reagents, Bruce and coworkers have shown applications to a diverse range of organisms such as Shewanella oneidensis [111], Escherichia coli [112 114], Pseudomonas aeruginosa [115], and even human cell lines [116]; other groups have reported similar concepts [117 119]. Recently, the group of Bruce also demonstrated that a quantitative dimension can be added to proteome wide crosslinking data with the help of metabolic stable isotope labeling [120].…”

Topographic Studies of the GroEL-GroES Chaperonin Complex by Chemical Cross-linking Using Diformyl Ethynylbenzene

“…However, these strategies tend not to simultaneously produce sequence ions that would identify the peptide. This leaves the identity of the peptides to be inferred by precursor mass alone (46,47) or requires non-standard instrumentation that permits multiple stages of dissociation (29). With electron transfer dissociation, DEB cross-linking not only produces sequence ions efficiently but also releases the individual molecular ions at equivalent intensity.…”

Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions

“…This can be achieved by the comparison of systematic interaction changes upon a specific stimuli or perturbation using cross-linking snapshots. Some pioneer studies have been applied in bacterial cells [8,10,24]. Another direction is quantitative crosslinking.…”

“…First and foremost, cross-linking can take a snap-shot of real-time dynamic interaction networks on the scale of the entire interactome in vitro and in vivo [8][9][10]. Also, cross-linking can not only identify the direct interacting partners but also determine the interaction sites at the same time.…”