Tandem Mass Spectrometry of Intact GroEL−Substrate Complexes Reveals Substrate-Specific Conformational Changes in the<i>trans</i>Ring

Duijn, Esther van; Simmons, Douglas A.; Heuvel, Robert H. H. van den; Bakkes, Patrick J.; Heerikhuizen, Harm van; Heeren, Ron M. A.; Robinson, Carol V.; Vies, Saskia M. van der; Heck, Albert J. R.

doi:10.1021/ja056756l

Cited by 89 publications

(92 citation statements)

References 52 publications

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“…Although the application of dissociative approaches to determining oligomeric stoichiometry is now well established, the use of CID to obtain details with respect to oligomeric organization is in its infancy. A growing base of evidence suggests that the ease of dissociation of the different constituent subunits might indicate whether they are located on the periphery or in the core of the complexes [57][58][59][60][61]. Validation of this hypothesis would allow confident inferences as to oligomeric topology from CID results, but a crucial twofold question remains to be answered: "In a heteromeric complex, what governs which subunits are ejected during CID and how does this relate to solution-phase topology?…”

Section: Frontiers In Gas-phase Activation Of Protein Complexesmentioning

confidence: 99%

Collisional activation of protein complexes: Picking up the pieces

Benesch

2009

J. Am. Soc. Mass Spectrom.

190

250

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Mass spectrometry is fast becoming a vital approach not only for the identification and quantification of proteins, but also for the study of the noncovalent assemblies they form. Approaches for ionizing, transmitting, and detecting protein complexes intact in the mass spectrometer are now well established. The challenge has therefore shifted to developing and applying mass spectrometry approaches to elucidate the structure of such species. A crucial aspect to this goal is inducing their disassembly in the gas phase to mine information as to their composition and organization. Here the consequences of collisionally activating protein complexes are illustrated through ion mobility mass spectrometry measurements and discussed in the context of the current literature. Although a consensus view of the mechanism of dissociation is starting to emerge, it is also clear that a number of aspects remain unresolved. These outstanding questions and frontier challenges must be addressed if gas-phase dissociative approaches are to reach their full potential in the study of protein assemblies. . Since that time technological and methodological developments have continued apace [2][3][4][5], such that the MS of such large species is no longer merely a technical curiosity, but rather a bona fide approach for structural biologists [6]. The many advantages that MS possesses, including speed and sensitivity of analysis [7], have made it integral to the fields of proteomics and systems biology [8,9]. The long-term challenge now is to extend the technology and methodology such that all higher levels of protein structure, from the secondary to the quinary [10], might be characterized rapidly and effectively by means of MS.Such a revolution will require the addition to, and adaptation of, the current conventional MS-based proteomic strategy. A cornerstone of this is tandem MS, wherein ions of interest are subjected to gas-phase dissociation and the fragments analyzed to provide protein sequence, and thus identity, information [11]. Recently much effort has been made to perform analogous experiments on protein complexes, whereby they are dissociated in the mass spectrometer to provide mass information on their constituents [12]. Furthermore, some evidence suggests that, aside from just providing the identity of subunits, the gas-phase dissociation process may even reveal details as to how these subunits are organized within the oligomer [13]. As such, the possibility has arisen that gas-phase dissociation coupled to MS might eventually allow the reverse engineering of protein assemblies.Over the last few years a considerable body of literature has emerged regarding the mechanism of the gas-phase dissociation of protein assemblies. A variety of activation techniques have been used [14 -18], but the most popular approach for the gas-phase dissociation of protein assemblies is currently collision-induced dissociation (CID). It is likely this is primarily attributable to its ease of implementation and its incorporation into the quadrupole t...

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Section: Frontiers In Gas-phase Activation Of Protein Complexesmentioning

confidence: 99%

Collisional activation of protein complexes: Picking up the pieces

Benesch

2009

J. Am. Soc. Mass Spectrom.

190

250

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“…The influence of ion size on the SID energytransfer efficiency, however, has been debated in the literature and is not entirely understood [24 -26], especially for systems as massive as noncovalent proteins. Nonetheless, collision with a surface should still be a more efficient process than collision with a gas for which efficiencies less than 0.01% per collision have been reported for large protein complexes [9]. SID of a few protein complexes has suggested that alternative ion activation methods may provide a way to extract meaningful structural data from the gas-phase fragmentation of protein complexes [27][28][29][30].…”

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confidence: 99%

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confidence: 99%

“…Mass spectrometry has recently provided insights on posttranslational modifications [1], mono-and polydisperse subunit stoichiometry [2], subunit organization [3,4], and noncovalent protein-ligand binding sites [5]. In addition to revealing structural information, mass spectrometry can be used to monitor dynamic processes, such as protein complex assembly [6], protein-substrate and protein-protein interactions [7,8], and substratespecific conformational changes [9].One of the limitations of current technology, however, is the fact that commercial instrumentation is still hampered by the amount of dissociation that can be induced from large biomolecular complexes. Often, MS has to be combined with many solution-based experiments (H/D exchange plus digestion, chemical crosslinking plus digestion, limited proteolysis, solution disruption by changes in ionic strength) because the MS instruments commercially available do not provide extensive dissociation of these massive complexes.…”

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confidence: 99%

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Surface-induced dissociation shows potential to be more informative than collision-induced dissociation for structural studies of large systems

Wysocki

Jones

Galhena

et al. 2008

J. Am. Soc. Mass Spectrom.

103

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The ability to preserve noncovalent, macromolecular assemblies intact in the gas phase has paved the way for mass spectrometry to characterize ions of increasing size and become a powerful tool in the field of structural biology. Tandem mass spectrometry experiments have the potential to expand the capabilities of this technique through the gas-phase dissociation of macromolecular complexes, but collisions with small gas atoms currently provide very limited fragmentation. One alternative for dissociating large ions is to collide them into a surface, a more massive target. Here, we demonstrate the ability and benefit of fragmenting large protein complexes and inorganic salt clusters by surface-induced dissociation (SID), which provides more extensive fragmentation of these systems and shows promise as an activation method for ions of increasing size. ver the past two to three decades, mass spectrometry (MS) has expanded significantly, from its early use as a technique for measuring the isotopes of elements and analyzing volatile compounds, to a technique that is now routinely used to study nonvolatile molecules and large macromolecular complexes. Increasingly, mass spectrometry and ion mobility/mass spectrometry are described as structural biology tools. Mass spectrometry has recently provided insights on posttranslational modifications [1], mono-and polydisperse subunit stoichiometry [2], subunit organization [3,4], and noncovalent protein-ligand binding sites [5]. In addition to revealing structural information, mass spectrometry can be used to monitor dynamic processes, such as protein complex assembly [6], protein-substrate and protein-protein interactions [7,8], and substratespecific conformational changes [9].One of the limitations of current technology, however, is the fact that commercial instrumentation is still hampered by the amount of dissociation that can be induced from large biomolecular complexes. Often, MS has to be combined with many solution-based experiments (H/D exchange plus digestion, chemical crosslinking plus digestion, limited proteolysis, solution disruption by changes in ionic strength) because the MS instruments commercially available do not provide extensive dissociation of these massive complexes. A typical dissociation result that is achieved is ejection of a monomer subunit as illustrated here (Figure 1) for a small heat shock protein (sHSP) dodecamer, consisting of 12 subunits each weighing 16.9 kDa [8,10,11]. The 16.9 kDa monomer typically carries away a large percentage of the charge of the original complex with the remainder of the charge on the 11-mer that weighs 186 kDa. Investigators have concluded that this behavior, which has been observed by several research groups for many different noncovalent protein complexes, happens because the low-energy collisions with a gas initiate unfolding of one of the protein subunits [12][13][14]. As this subunit unfolds, it gains surface area, thus allowing it to accept more charge. Eventually, when the unfolding protein and the remainde...

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“…Analysis of large MDa complexes such as the ribosome 19 and highly ordered virus capsids [20][21][22] , in defining substrate binding to molecular machines [23][24][25] , or the characterization of subunit interaction networks 26,17,16,17,27,28 serve as but a few examples of the value of this approach.…”

Section: Discussionmentioning

confidence: 99%

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Kirshenbaum

Michaelevski

Sharon

2010

JoVE

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Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes 1 . To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multiprotein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS) [2][3][4][5] . This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture.Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

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Tandem Mass Spectrometry of Intact GroEL−Substrate Complexes Reveals Substrate-Specific Conformational Changes in thetransRing

Cited by 89 publications

References 52 publications

Collisional activation of protein complexes: Picking up the pieces

Collisional activation of protein complexes: Picking up the pieces

Surface-induced dissociation shows potential to be more informative than collision-induced dissociation for structural studies of large systems

Analyzing Large Protein Complexes by Structural Mass Spectrometry

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