Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets

Han, Linjie; Ruotolo, Brandon T.

doi:10.1021/acs.analchem.5b01010

Cited by 21 publications

(13 citation statements)

References 33 publications

(88 reference statements)

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“…By following the gas phase unfolding pattern of protein ions, the unfolding “heat maps” [41] are a function of protein higher order structure and protein-ligand binding state [39, 42, 53–55]. Both intact OCP and NTD undergo gas phase unfolding to increase their CCS, whereas CTD remarkably undergoes no significant unfolding (see Figure 5 for maps of intact OCP monomer, dimer, OCP NTD and CTD).…”

Section: Resultsmentioning

confidence: 99%

Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains

Zhang¹,

Liu²,

Lü³

et al. 2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD) and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes.

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Section: Resultsmentioning

confidence: 99%

Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains

Zhang¹,

Liu²,

Lü³

et al. 2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…GD dodecamer is a nonspecific dimer of the GD hexamer complex (337 kDa). In contrast to other nonspecific adducts of protein complexes, e.g., avidin octamer (Figure S2B) or GroEL 28mer, 54 GD dodecamer dissociates symmetrically into the constituent hexamers under both IRMPD and CID, 63 while the asymmetric dissociation of hexamers is hindered (Figure S5).…”

Section: Analytical Chemistrymentioning

confidence: 99%

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase

et al. 2016

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Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment.

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“…According to our MD simulations, three out of the six isoforms appear to be energetically favorable for doubly charged W4 with, however, only two of them forming the more hairpin-line structure with the above-mentioned salt bridge. Like in comparable systems, [11] the absolute values of computed CCSs and measured DT CCSs He differ depending on the method, but the coexistence of distinct species is obvious from MD simulations and experimental ATDs alike (Figure S5) and has also been observed with related, singly charged peptides. [12]…”

Section: Resultsmentioning

confidence: 96%

Conformational Shift of a β‐Hairpin Peptide upon Complex Formation with an Oligo–proline Peptide Studied by Mass Spectrometry

et al. 2016

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So-called super-secondary structures such as the β-hairpin, studied here, form an intermediate hierarchy between secondary and tertiary structures of proteins. Their sequence-derived ‘pure’ peptide backbone conformation is combined with ‘remote’ interstrand or interresidue contacts reminiscent of the 3D-structure of full-length proteins. This renders them ideally suited for studying potential nucleation sites of protein folding reactions as well as intermolecular interactions. But β-hairpins do not merely serve as model systems; their unique structure characteristics warrant a central role in structural studies on their own. In this study we applied photo cross-linking in combination with high-resolution mass spectrometry and computational modeling as well as with ion mobility-mass spectrometry to elucidate these structural properties. Using variants of a known β-hairpin representative, the so-called trpzip peptide and its ligands, we found evidence for a conformational transition of the β-hairpin and its impact on ligand binding.

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Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets

Cited by 21 publications

References 33 publications

Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains

Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains

Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase

Conformational Shift of a β‐Hairpin Peptide upon Complex Formation with an Oligo–proline Peptide Studied by Mass Spectrometry

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