Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures

Tian, Yuwei; Han, Linjie; Buckner, Adam C.; Ruotolo, Brandon T.

doi:10.1021/acs.analchem.5b03291

Cited by 157 publications

(263 citation statements)

References 43 publications

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“…We found that engineered covalent links between core and dimer domain restricts unfolding of NapA. Notably, the introduction of a disulphide bond in of itself does not restrict gas-phase unfolding in other protein systems25, implying that the covalent linkages used here are influencing specific unfolding events in NapA. These restricted unfolding results imply that the core domains in the native protein are almost certainly flexibly attached, and these interactions are disrupted during gas-phase activation.…”

Section: Discussionmentioning

confidence: 80%

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

et al. 2017

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Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.

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Section: Discussionmentioning

confidence: 80%

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

et al. 2017

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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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“…For example, ion mobility (IM) can be coupled with MS to obtain collision cross sections, yielding information about overall molecular shape [3][4][5][6][7][8]. Collision induced unfolding has recently proven to be valuable in distinguishing antibody isoforms by comparing "fingerprint plots" generated by differences in unfolding of the isoforms in an ion trap prior to IM-MS analysis [9]. Ion-ion reactions can be used to assign charge states in very large systems [10].…”

mentioning

confidence: 99%

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

Bonner

Hendricks

Julian

2016

J. Am. Soc. Mass Spectrom.

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Abstract. Significant effort is being employed to utilize the inherent speed and sensitivity of mass spectrometry for rapid structural determination of proteins; however, a thorough understanding of factors influencing the transition from solution to gas phase is critical for correct interpretation of the results from such experiments. It was previously shown that combined use of action excitation energy transfer (EET) and simulated annealing can reveal detailed structural information about gaseous peptide ions. Herein, we utilize this method to study microsolvation of charged groups by retention of 18-crown-6 (18C6) in the gas phase. In the case of GTP (CEGNVRVSRE LAGHTGY), solvation of the 2+ charge state leads to reduced EET, whereas the opposite result is obtained for the 3+ ion. For the mini-protein CTrpcage, solvation by 18C6 leads to dramatic increase in EET for the 3+ ion. Examination of structural details probed by molecular dynamics calculations illustrate that solvation by 18C6 alleviates the tendency of charged side chains to seek intramolecular solvation, potentially preserving native-like structures in the gas phase. These results suggest that microsolvation may be an important tool for facilitating examination of native-like protein structures in gas phase experiments.

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Collision Induced Unfolding of Intact Antibodies: Rapid Characterization of Disulfide Bonding Patterns, Glycosylation, and Structures

Cited by 157 publications

References 43 publications

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

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

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization

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