Thermal Decomposition of a Gaseous Multiprotein Complex Studied by Blackbody Infrared Radiative Dissociation. Investigating the Origin of the Asymmetric Dissociation Behavior

Felitsyn, Natalia; Kitova, Elena N.; Klassen, John S.

doi:10.1021/ac0103975

Cited by 174 publications

(304 citation statements)

References 65 publications

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“…For example, in the gas phase the tetrameric assemblies of adult human hemoglobin and concanavalin dissociate via the loss of a single subunit [6], while in solution they are formed by the association of two specific dimers, rather than by the association of individual subunits. Similar asymmetric dissociation behavior is reported in a recent study [7] of the thermal decomposition of a gaseous multiply protonated ho- …”

supporting

confidence: 88%

“…The experimental apparatus and procedures used in this work have been described in detail elsewhere [7] and only a brief overview is given here. All experiments were performed on an ApexII 47e Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker, Billerica, MA) equipped with a modified external nanoelectrospray ion source.…”

Section: Methodsmentioning

confidence: 99%

“…If proton transfer does occur, then the mobility of the protons is expected to be sensitive to the internal energy of the ions. In the recent BIRD study of the B 5 pentamer, it was shown that the degree of charge enrichment of the subunit that was lost was sensitive to the reaction temperature, with higher temperature favoring enrichment [7]. Based on this result it was concluded that at least some of the protons were able to migrate in the gas phase between the subunits and account for the observed enrichment of charge on the leaving subunit.…”

mentioning

confidence: 99%

“…The use of multiple stages of MS with ion activation/dissociation (MS n ) to dissect gaseous assemblies into their constituent subunits represents an attractive, yet unproven, strategy for determining subunit composition and topology. In recent years, a number of MS n studies of gaseous protein dimers [4,5], tetramers [6], and pentamers [7] have been reported. Homo-and heterodimers are readily decomposed into their individual subunits, most generally by collision-induced dissociation (CID).…”

mentioning

confidence: 99%

“…Two salt bridges between NH1 of C-terminal Arg 142 and OE1 of N-terminal Glu 2 further stabilize the dimer in solution. In the gas phase, hydrogen bonds are expected to be the dominant interactions stabilizing the dimer [7,14]. The time-resolved BIRD experiments were used to determine the temperature dependence of the rate constants for the dissociation of the dimer into monomers and thereby the Arrhenius activation parameters.…”

mentioning

confidence: 99%

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Thermal dissociation of the protein homodimer ecotin in the gas phase

Felitsyn

Kitova

Klassen

2002

J. Am. Soc. Mass Spectrom.

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The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique. Dissociation of the protonated dimer, (E 2 ϩ nH) nϩ § E 2 nϩ where n ϭ 14 -17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175°C. The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50 -60% of the monomer product ions correspond to an asymmetric partitioning of charge. The relative abundance of the different monomer ion pairs produced from E 2 14ϩ , E 2 15ϩ , and E 2 16ϩ depends on reaction time, with the more symmetric charge distribution pair dominating at longer times. The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers. For E 2 17ϩ , the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution. The charge distribution in the E 2 15ϩ and E 2 16ϩ dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity. In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E 2 17ϩ dimer. (J Am Soc Mass Spectrom 2002, 13, 1432-1442) © 2002 American Society for Mass Spectrometry P rotein complexes composed of two or more subunits are believed to constitute the bulk of soluble and membrane-bound proteins [1,2]. While the importance of protein assemblies in cellular function is well recognized, their structural characterization remains a significant analytical challenge. Mass spectrometry (MS), with its speed, sensitivity, and accurate mass capability holds tremendous potential as a tool for characterizing protein assemblies [3]. The use of multiple stages of MS with ion activation/dissociation (MS n ) to dissect gaseous assemblies into their constituent subunits represents an attractive, yet unproven, strategy for determining subunit composition and topology. In recent years, a number of MS n studies of gaseous protein dimers [4,5], tetramers [6], and pentamers [7] have been reported. Homo-and heterodimers are readily decomposed into their individual subunits, most generally by collision-induced dissociation (CID). An interesting feature of the dissociation behavior of the protein-protein complexes is the tendency for uneven sharing of charge between subunits, even in the case of homodimers. For example, the decomposition of four homodimers (human galectin I, E. coli glyoxalase I, horse heart cytochrome c, and hen egg lysozyme) yields a highly asymmetric charge distribution, with one of the subu...

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supporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Thermal dissociation of the protein homodimer ecotin in the gas phase

Felitsyn

Kitova

Klassen

2002

J. Am. Soc. Mass Spectrom.

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Ion Activation Methods for Tandem Mass Spectrometry

Håkansson

Klassen

2010

Electrospray and MALDI Mass Spectrometry

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Ion Mobility–Mass Spectrometry Reveals Long‐Lived, Unfolded Intermediates in the Dissociation of Protein Complexes

et al. 2007

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Recent applications of mass spectrometry (MS) in structural biology have highlighted its ability to define the stoichiometry of numerous protein complexes. [1,2] When combined with tandem MS, this extra dimension has made possible 1) analysis of polydisperse assemblies, [3] 2) characterization of release of proteins from within proteasome and chaperone complexes, [4,5] and 3) identification of proteins released from intact megadalton ribosomes.[6] Tandem MS is effective because macromolecular protein-complex ions decay through a mechanism that involves a dramatic transfer of charge to monomeric subunits prior to their ejection. This asymmetric dissociation acts to distribute the product ion spectrum over a large m/z range, thus enabling separation of ions with overlapping m/z values and identification of heterocomplexes from released subunits.It has been proposed that protein unfolding events are involved in the dissociation mechanism, [7][8][9] although direct evidence pertaining to the structure of the intermediates has not been reported. Ion mobility (IM)-MS, [10,11] a gas-phase technology that separates ions based on their size and shape, has been used to explore gas-phase protein folding, [12][13][14] oligonucleotide structures, [15] and noncovalent complexes. [16][17][18] Herein, we apply IM-MS to examine the activated form of a macromolecular complex. For our studies, we used the 56-kDa complex of tetrameric transthyretin (TTR), primarily because its gas-phase dissociation behavior has been studied extensively.[19] Our experiments were performed on a quadropole-ion mobility-time of flight (Q-IM-ToF) instrument (Synapt, Waters, Milford MA, USA, see the Experimental Section) using an IM separator that employs a series of low-voltage DC waves to push ions through a drift chamber filled with neutral molecules (0.5-1 mBar N 2 ).[20] The speed with which an ion traverses the drift region depends on its collision cross section (CCS); ions with larger CCSs proceed more slowly than ions with smaller ones.These drift times are then calibrated using protein ions of known CCS.[18] Subsequently, molecular modeling is used to generate a range of possible structures, and the CCSs for these models are calculated for comparison with experimental values.Mass spectra for TTR recorded under conditions designed either to maintain or to activate the intact oligomers (80 V or 150 V accelerating voltage, respectively, in the source region of the instrument) are shown (Figure 1 A, C). At 80 V, the minimum accelerating voltage for observation of massresolved protein-complex ions, peaks in the spectrum can be assigned to TTR tetramer and octamer. At 150 V, peaks corresponding to the octamer and tetramer persist; however, under these conditions monomeric subunits are also evident (Figure 1 C). This result indicates that a population of oligomeric TTR ions is undergoing the initial stages of dissociation and is therefore at an ideal stage for analysis of activated states of the complexes.IM data shows that without activation, both tet...

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Thermal Decomposition of a Gaseous Multiprotein Complex Studied by Blackbody Infrared Radiative Dissociation. Investigating the Origin of the Asymmetric Dissociation Behavior

Cited by 174 publications

References 65 publications

Thermal dissociation of the protein homodimer ecotin in the gas phase

Thermal dissociation of the protein homodimer ecotin in the gas phase

Ion Activation Methods for Tandem Mass Spectrometry

Ion Mobility–Mass Spectrometry Reveals Long‐Lived, Unfolded Intermediates in the Dissociation of Protein Complexes

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