Characterisation of the heptameric pore‐forming complex of the <i>Aeromonas</i> toxin aerolysin using MALDI‐TOF mass spectrometry

Moniatte, Marc; Goot, F. Gisou van der; Buckley, J. Thomas; Pattus, Franc; Dorsselaer, Alain Van

doi:10.1016/0014-5793(96)00328-6

Cited by 97 publications

(69 citation statements)

References 36 publications

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“…Except in cases where the association constant is extremely high [30], MALDI mass spectrometry is not suitable for observation of noncovalently bound species. In order to observe the oligomeric forms of HSP90, EDC was used to cross-link interacting species prior to MALDI mass spectrometric analysis.…”

Section: Mass Spectrometry Of Oligomeric Speciesmentioning

confidence: 99%

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

Garnier

Lafitte

Jørgensen

et al. 2001

European Journal of Biochemistry

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HSP90 is one of the most abundant proteins in the cytosol of eukaryotic cells. HSP90 forms transient or stable complexes with several key proteins involved in signal transduction including protooncogenic protein kinases and nuclear receptors, it interacts with cellular structural elements such as actin-microfilament, tubulin-microtubule and intermediate filaments, and also exhibits conventional chaperone functions. This protein exists in two isoforms a-HSP90 and b-HSP90, and it forms dimers which are crucial species for its biological activity. PAGE, ESI-MS and MALDI-MS were used to study HSP90 purified from pig brain. The two protein isoforms were clearly distinguished by ESI-MS, the a isoform being < six times more abundant than the b isoform. ESI-MS in combination with l phosphatase treatment provided direct evidence of the existence of four phosphorylated forms of native pig brain a-HSP90, with the diphosphorylated form being the most abundant. For the b isoform, the di-phosphorylated was also the most abundant. MALDI mass spectra of HSP90 samples after chemical cross-linking showed a high percentage of a±a homodimers. In addition, evidence for the existence of higher HSP90 oligomers was obtained.Keywords: HSP90; mass spectrometry; phosphorylation; cross-linking.Heat shock proteins (HSP) are abundant eukaryotic proteins constituting < 1 or 2% of the cellular proteins under physiological conditions, with an increased expression in response to stress [1]. These ubiquitous proteins, classified according to their apparent molecular mass, i.e. HSP27, 60, 70 or 90, work in close association with cytosolic cofactors to help the correct folding of several cytosolic proteins (reviewed in [2]).The target of this study is HSP90, a dimeric phosphoprotein involved in many cell processes such as cell cycle control, transcriptional regulation or signal transduction (reviewed in [3]). Mammalian HSP90 exists in two isoforms a-and b-HSP90 [4], differing in mass by < 2 kDa. Although dimers appear to be the functional form of HSP90, oligomeric forms exist in solution under physiological conditions [5] and their amount tends to increase under hyperthermal exposure [6±9]. HSP90 possesses an ATP binding site [10,11] and undergoes conformational changes in presence of ATP [12,13]. This protein also possesses ATPase activity [14]. The functions of HSP90 are in part regulated by phosphorylation, e.g. phosphorylated HSP90 stimulates eIF-2a kinase activity [15] and phosphorylation regulates the interaction between HSP90 and pp60v-src [16]. HSP phosphorylation may be mediated by various kinases [17,18] but this protein can also autophosphorylate and act as a kinase for other protein substrates [19].ESI-MS and MALDI-MS are the most suitable mass spectrometric techniques for the analysis of macromolecules [20,21]. The increase in resolution achieved by the latest generation of mass spectrometers allows a detailed investigation of post-translational modifications of large proteins and protein complexes [22]. This is particularly important ...

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Section: Mass Spectrometry Of Oligomeric Speciesmentioning

confidence: 99%

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

Garnier

Lafitte

Jørgensen

et al. 2001

European Journal of Biochemistry

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“…Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower highmass sensitivity of MCP detectors. Several major effects have been described to result in nonnative conditions for the noncovalent complexes: MALDI matrix [3][4][5], sample preparation [4,6,7], crystal morphology [4,8,9], pH of the solution [6, 10 -12], organic solvent [7,8], ionic strength [13,14], matrix/analyte ratio [15,16], speed of solvent evaporation [4], and sample concentration [9,15]. The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.…”

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confidence: 99%

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

Yanes

Aviles

Roepstorff

et al. 2007

J. Am. Soc. Mass Spectrom.

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The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-M range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower highmass sensitivity of MCP detectors. Several major effects have been described to result in nonnative conditions for the noncovalent complexes: MALDI matrix [3][4][5], sample preparation [4,6,7], crystal morphology [4,8,9], pH of the solution [6, 10 -12], organic solvent [7,8], ionic strength [13,14], matrix/analyte ratio [15,16], speed of solvent evaporation [4], and sample concentration [9,15]. The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.Also, analyses by electrospray ionization (ESI) [21] are not carried out at physiological conditions as only solutions of very low ionic strength can be analyzed. However, ESI generates "colder" ions than MALDI and it keeps the sample in aqueous "biological" environment before ionization. For these reasons, ESI has been used in numerous studies to detect noncovalent complexes [22].Relatively few cases have been reported where specific intact noncovalent complexes were successfully observed with MALDI. Besides the frequent dissociation of noncovalent complexes due to the experimental conditions employed, a further...

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“…This work showed the feasibility of directly measuring hydrogen/deuterium exchange kinetics of detergent-solubilized proteins in solution by ES mass spectrometry and thereby obtaining structural information. Although ES has now been extensively used in the direct study of noncovalent interactions, the application of matrix-assisted laser desorption ionization (MALDI) in this area of research been more restricted, although it should be noted that Van Dorsselaer et al (33,34) have reported MALDI data on the subunit stoichiometry of high mass noncovalent complexes of membrane proteins.…”

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confidence: 99%

Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

Lengqvist

Svensson

Evergren

et al. 2004

Journal of Biological Chemistry

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Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer⅐glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys 49 (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144 -15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.Membrane proteins are estimated to constitute about onethird of the total proteome (1) and have important and diverse roles in biology, including functions as cell-surface receptors, ion channels, and transporters and in cell adhesion. However, because of the difficulty in producing crystals of detergentsolubilized proteins for x-ray crystallography, only ϳ1% of the unique protein structures so far solved at high resolution are of membrane proteins (2). Integral membrane proteins thus pose an important challenge in structural biology.Microsomal glutathione transferase-1 (MGST1) 1 is a member of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily. At present, other mammalian members of the MAPEG family are two glutathione transferases/peroxidases (MGST2 and MGST3), 5-lipoxygenase-activating protein, leukotriene C 4 synthase, and microsomal prostaglandin E 2 synthase (the closest relative to MGST1) (3). MGST1 functions both as a glutathione transferase and as a peroxidase, thus potentially protecting cells from reactive compounds and oxidative stress (3, 4). The quaternary structure of MGST1 has previously been investigated by electron crystallography (5, 6), radiation inactivation (7), cross-linking (8), and hydrodynamic (9) studies and has been suggested to be a homotrimer both in the microsomal membrane and in the purified form. Electrospray (ES) mass spectrometry has shown that the enzyme is partly N-acetylated (10). Interestingly, equilibrium dialysis (11) and stopped-flow active-site titrations (12) have indicated that the MGST1 homotrimer binds only one molecule of ...

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Characterisation of the heptameric pore‐forming complex of the Aeromonas toxin aerolysin using MALDI‐TOF mass spectrometry

Cited by 97 publications

References 36 publications

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

Phosphorylation and oligomerization states of native pig brain HSP90 studied by mass spectrometry

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

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