Observation of the Noncovalent Quaternary Associations of Proteins by Electrospray Ionization Mass Spectrometry

Light-Wahl, Karen J.; Schwartz, Brenda L.; Smith, Richard D.

doi:10.1021/ja00091a035

Cited by 281 publications

(308 citation statements)

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“…However, once these conditions were found, they were generally applicable for both the XL and Centaur instruments, with some minor adjustments needed between experiments due to spraying variation. As a control, both concanavalin A and hemoglobin were sprayed in order to confirm the optimized instrument parameters and trends noted herein; in both cases a majority of tetrameric species was observed (data not shown) in accordance with previous observations [21,22]. It was quickly discovered that increasing the q2 pressure in the Centaur beyond its original limit did not offer any advantage.…”

Section: General Observations For Neutral Inos Solutionssupporting

confidence: 79%

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Smith

Siu

Rafferty

2004

J. Am. Soc. Mass Spectrom.

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The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H 4 B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H 4 B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 3 Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions. (J Am Soc Mass Spectrom 2004, 15, 629 -638)

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Section: General Observations For Neutral Inos Solutionssupporting

confidence: 79%

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Smith

Siu

Rafferty

2004

J. Am. Soc. Mass Spectrom.

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“…In order to evaluate the stability of the c-Myb DBDdsDNA complexes with different solution-phase Kd MBS-I 16 4 (ϩ) 5= CCT AAC TGA CAC ACA T 3= -3.8 ϫ 10 Ϫ9 (Ϫ) 3= GGA TTG ACT GTG TGT A 5= m22 4 (ϩ) 5= CAC CCT AAC TGA CAC ACA TTC T 3= 0 2.8 ϫ 10 Ϫ9 (Ϫ) 3= GTG GGA TTG ACT GTG TGT AAG A 5= m3 5 (ϩ) 5= CAC CCT CAC TGA CAC ACA TTC T 3= Ϫ2.9 3.8 ϫ 10 Ϫ7 (Ϫ) 3= GTG GGA GTG ACT GTG TGT AAG A 5= m4 5 (ϩ) 5= CAC CCT ATC TGA CAC ACA TTC T 3= Ϫ1.5 3.6 ϫ 10 Ϫ8 (Ϫ) 3= GTG GGA TAG ACT GTG TGT AAG A 5= m5 5 (ϩ) 5= CAC CCT AGC TGA CAC ACA TTC T 3= Ϫ2.0 8.3 ϫ 10 Ϫ8 (Ϫ) 3= GTG GGA TCG ACT GTG TGT AAG A 5= m6 5 (ϩ) 5= CAC CCT ACC TGA CAC ACA TTC T 3= Ϫ1.4 3.0 ϫ 10 Ϫ8 (Ϫ) 3= GTG GGA TGG ACT GTG TGT AAG A 5= m14 5 (ϩ) 5= CAC CCT AAC TCA CAC ACA TTC T 3= Ϫ3.2 6.3 ϫ 10 Ϫ7 (Ϫ) 3= GTG GGA TTG AGT GTG TGT AAG A 5= m19 5 (ϩ) 5= CAC CCT AAC TGA TAC ACA TTC T 3= Ϫ1.0 1.5 ϫ 10 Ϫ8 (Ϫ) 3= GTG GGA TTG ACT ATG TGT AAG A 5=…”

Section: Characterization Of the Complex Stability By Cidmentioning

confidence: 99%

“…The DNA and c-Myb DBD was incubated in buffer (100mM potassium phosphate (pH7.5), 20mM KCl, 0.1mM EDTA, 500 g/mL of BSA, 5% glycerol (v/v), 10mM dithiothreitol) on ice, and then filtered through nitrocellulose under suction [11]. 4 Sequences of MBS-1 16 and m22 are wild-type. 5 m3, m4, m5, m6, m14, and m19 are single point mutants of m22.…”

Section: Characterization Of the Complex Stability By Cidmentioning

confidence: 99%

“…Consequently, positive correlation was obtained between V 50% and relative binding free energy change (⌬⌬G) [1]. In addition, ESI-MS has allowed successful detection of noncovalent complexes of biomolecules [2][3][4][5][6]. Direct observation of molecular ions of noncovalent complexes is significant in understanding protein function, because it suggests a target, specificity, and stoichiometry of the specific binding.…”

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confidence: 99%

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Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

Akashi

Osawa

Nishimura

2005

J. Am. Soc. Mass Spectrom.

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Binding affinity of complexes between a DNA-binding domain (DBD) of a transcription factor, c-Myb, and several double-stranded DNA (dsDNA) were evaluated by collision-induced dissociation (CID) of the multiply protonated molecules generated by electrospray ionization mass spectrometry (ESI-MS). Complexes of the c-Myb DBD and dsDNA were prepared in solution and analyzed by ESI-MS. Multiply protonated molecules of a high-affinity complex, the c-Myb DBD and dsDNA with a specific sequence, were clearly observed in ESI mass spectrum. Protonated molecules of the complex were quite stable in the gas-phase, and not easily dissociated even if high cone voltage was applied in the first vacuum chamber source when the sample was prepared in 10 mM ammonium acetate. As for the sample prepared in buffer with higher concentration of ammonium acetate, such as 500 mM ammonium acetate, protein-dsDNA complexes could easily be dissociated with an increase in the cone voltage, giving multiply protonated molecules of free c-Myb DBD and some DNA fragments. Systematic CID experiments were carried out on seven complexes between the c-Myb DBD and 22-mer dsDNA with different solution-Kd values in the range of 10 Ϫ9 M to 10 Ϫ7 M. For each complex dissociation curve as a function of cone voltage was plotted, and the cone voltage where 50% of the complex was dissociated (V 50% ) was calculated. Consequently, positive correlation was obtained between V 50% and relative binding free energy change (⌬⌬G) in complex formation in solution. This suggests that ESI-CID experiments can provide quantitative evaluation of the stability of protein-DNA complexes based on proper calibration. (J Am Soc Mass Spectrom 2005, 16, 116 -125)

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“…It is not disrupted by guanidine hydrochloride, pH extremes, SDS, reducing agents or heat and it is resistant to many proteases [14]. Electrospray ionization mass spectrometry (ESMS) [15] has previously been used to characterise non-covalent receptor-ligand complexes [16], oligonucleotide associations [17,18], non-covalent protein-peptide complexes [19] and more recently to study the tetramers formed by avidin [20], streptavidin [21], hemoglobin [20] as well as yeast alcohol dehydrogenase and rabbit muscle pyruvate kinase [22]. However, the use of ESMS is usually limited to mass measurements below 150 kDa due to the difficulty of resolving multiply charged ions at higher mass.…”

Section: Introductionmentioning

confidence: 99%

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

Moniatte¹,

Goot

Buckley

et al. 1996

FEBS Letters

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Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of ~-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-24631. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a beptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins.

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Observation of the Noncovalent Quaternary Associations of Proteins by Electrospray Ionization Mass Spectrometry

Cited by 281 publications

References 0 publications

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Evaluation of protein-DNA binding affinity by electrospray ionization mass spectrometry

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

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