An atomic structure of the full 26 S, however, has yet to be reported, presumably because of its heterogeneity and inherent dynamics. modifications. As such biological MS has evolved from being a purely auxiliary technique, used for the analysis of single peptide fragments for protein identification, to a major driving force in large scale proteomics and modern systems biology (14). In parallel with these developments, MS is also emerging as a powerful approach for determining protein-protein interactions, subunit composition (2,59), and architectural organization of intact protein complexes (5,1013). Protein-protein interactions have traditionally been investigated by affinity purification coupled with MS (AP-MS).1AP-MS uses affinity tags to the proteins of interest or antibodies against to pull down the protein of interest and associated interactors, which are then analyzed and identified by proteomics-based MS (2,5,14). Using AP-MS, it is therefore possible to investigate the connectivity within a complex Mouse monoclonal to 4E-BP1 or even establish whole protein interaction networks. By contrast, MS of intact assemblies usually focuses on a single protein complex or at least a relatively well defined assembly. However, for large heterogeneous complexes, MS of intact complexes is then capable of elucidating not only connectivity and mass but to also provide key information on absolute subunit stoichiometry, heterogeneity, and dynamical changes. The discovery in the early 1990s that noncovalent interactions could be maintained intact in the gas phase by the use of ESI (15) and the introduction of specialized instrumentation (16) and reliable protocols (17) enabled the use of MS in the study of intact complexes. These advancements paved the way to an area of research known as Ganirelix native MS or MS of intact protein assemblies (for general reviews, see Refs.10,11,13, and18). The initial challenges in the Ganirelix analysis of macromolecular protein complexes by MS involved finding conditions for maintaining interactions. The protein complex, made up of multiple protein subunits and kept together by weak, noncovalent interactions, must survive the desolvation and ionization processes. Following these steps, the complex must be transferred intact into the gas phase. Finally, it has to pass through the instrument without dissociation and impinge upon the detector with sufficient impact for detection. Additionally, controlled dissociation is often required to elucidate fully the overall composition of the intact complex. Many of these initial challenges have now been overcome. Briefly, gentle ionization takes place at atmospheric pressure, typically via nanoflow electrospray from buffered aqueous solutions to preserve the noncovalent interactions of the intact assembly (17). Ionized protein complexes, encapsulated in small droplets, then experience a free jet expansion during their traversal to the rough vacuum of the instrument. Here, optimization of instrument conditions, namely pressure and accelerating voltages, is required to transfer the protein complex of interest intact into the gas phase. This allows for its traversal through the instrument without dissociation. Because of their high impact, very large ions can reach kinetic energies of tens to hundreds of electron volts and consequently become defocused (19). Moreover, because of their very high kinetic energies, these ions cannot be focused by typical ion optics. The passage of protein complexes through the mass spectrometer therefore requires higher pressures in the instrument than one would normally use, in a process known as collisional cooling or focusing (20,21). This results in collisions between large ions and small neutral gas molecules that now convert some of the kinetic energy into internal energy and help to focus the ion beam (19). Having maintained all the interactions within the complex, an initial spectrum of the intact assembly is then recorded. This provides information on the overall subunit stoichiometry and homogeneity. Controlled activation and dissociation of the intact complex is then carried out. This permits interrogation of subcomplexes and protein-protein interactions, either through gas phase dissociation or solution-based disruption. Numerous techniques for controlled gas phase dissociation of protein complexes have been developed (22,23). In collision-induced dissociation, the most commonly applied, dissociation is achieved by subjecting the protein complex to collisions Ganirelix with an inert gas within a collision cell. The exact mechanism of dissociation is the subject of much ongoing research (2427) and is only discussed briefly here. From the study of a large variety of protein complexes, a predominant dissociation pathway has started to emerge, although exceptions have also been found. An increase in internal energy caused by collision events enables.