Stabilization of Gas-Phase Noncovalent Macromolecular Complexes in Electrospray Mass Spectrometry Using Aqueous Triethylammonium Bicarbonate Buffer

Lemaire, David; Marie, Gérald; Sérani, Laurent; Laprévôte, Olivier

doi:10.1021/ac001276s

Cited by 79 publications

(79 citation statements)

References 29 publications

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“…Changing the nature of the buffer salt produced little change to the spectra, except that, with everything else being equal, there was a shift towards lower charge states, when triethylammonium bicarbonate was used. This shift is in line with the higher proton affinity of triethylammonia than that of ammonia; the former is expected to be much more efficient than the latter in competing with iNOS for the protons [44]. Furthermore, raising the solution pH from 7 through 10 appeared also to have little effect on the spectra.…”

Section: General Observations For Neutral Inos Solutionsmentioning

confidence: 54%

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 Solutionsmentioning

confidence: 54%

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 solvent for all samples was water. NH 4 AcO and (HNEt 3 )HCO 3 were used to buffer the proteininhibitor solutions; these are well known for conserving noncovalent interactions in ESI [37]. The titration samples contained between 8.8 and 9.0 M AK (depending on the volume of added inhibitor), 0 to 9.6 M P 1 ,P 5 -di(adenosine-5')pentaphosphate (Ap5A) or 0 to 12 M P 1 ,P 4 -di(adenosine-5')tetraphosphate (Ap4A), 50mM (HNEt 3 )HCO 3 , and 500 M EDTA.…”

Section: Methodsmentioning

confidence: 99%

Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors

Daniel

McCombie

Wendt

et al. 2003

J. Am. Soc. Mass Spectrom.

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Noncovalent complexes between chicken muscle adenylate kinase and two inhibitors, P 1 ,P 4 -di(adenosine-5')tetraphosphate (Ap4A) and P 1 ,P 5 -di(adenosine-5') pentaphosphate (Ap5A), were investigated with electrospray ionization mass spectrometry under non-denaturing conditions. The nonconvalent nature and the specificity of the complexes are demonstrated with a number of control experiments. Titration experiments allowed the association constants for inhibitor binding to be determined. Problems with concentration dependent ion yields are circumvented by a data evaluation method that is insensitive to the overall ionization efficiency. The K a values found were 9.0 ϫ 10 4 M Ϫ1 (Ap4A) and 4.0 ϫ S oft ionization mass spectrometry (MS), in particular matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), is well known for its ability to bring high molecular weight biomolecules into the gase phase and to even preserve noncovalent complexes [1][2][3][4][5][6][7][8][9][10]. Besides its established applications, a recurring question concerns the applicability of mass spectrometry to measuring noncovalent interaction strengths [11]. There are different approaches to measure the interaction strengths in the gas phase as well as in the liquid phase. In the former case, the mass spectra should ideally directly represent the solution phase chemistry. Gas-phase methods include cone voltage driven dissociation (called the "VC 50 " method) [12,13], collision-induced dissociation [14,15], guided ion beam tandem mass spectrometry [16,17], blackbody infrared radiative dissociation [18,19] and heated capillary dissocation [20,21].Some mass spectrometric studies have been reported in the literature for the determination of noncovalent interaction strengths. There are three basic methods to determine association constants that are reflective of those in solution: first, it is possible to record "melting curves" by raising the temperature of the analyte solution and to use MS to determine the percentage of intact complexes. Cheng et al. analyzed complementary DNA strands [22] and Fändrich et al. studied the chaperone GimC/prefolding homologue complex [13] with this method. Second, competition experiments can be used to evaluate the stability of noncovalent complexes. In Jørgensen's method [23,24], the relative intensities of the free host and the different complexes were used, whereas Kempen et al. employed the known concentration of a reference complex to determine the concentration of an unknown complex [25]. Third, a titration of a host with a guest (or vice versa) can be used. In this case, the mass spectrometric signals should accurately represent the concentrations of the species in solution. The titration method is documented in a number of publications, all of them convincingly showing the successful use of mass spectrometry for the quantitative determination of noncovalent binding constants of proteins and small molecules [26 -33]. This aspect was the main focus in most previous research; th...

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“…The model proposed by de la Mora and Felitsyn also predicts that when other than ammonium acetate buffer salts are used this may influence the charging of globular proteins and protein complexes. It has been shown that triethylammonium bicarbonate can also be used in ESI mass spectrometry for studying intact proteins and non-covalent complexes (Lemaire et al, 2001). In comparison with ammonium bicarbonate or acetate solutions, the use of triethylammonium bicarbonate resulted in significantly less ion charges, which may be explained by the higher gas-phase proton affinity of protonated triethylammonium bicarbonate (Lemaire et al, 2001).…”

Section: Electrospray Ionization Of Biomacromoleculesmentioning

confidence: 99%

“…It has been shown that triethylammonium bicarbonate can also be used in ESI mass spectrometry for studying intact proteins and non-covalent complexes (Lemaire et al, 2001). In comparison with ammonium bicarbonate or acetate solutions, the use of triethylammonium bicarbonate resulted in significantly less ion charges, which may be explained by the higher gas-phase proton affinity of protonated triethylammonium bicarbonate (Lemaire et al, 2001). Moreover, it has been proposed that the multiply charged ions generated in a triethylammonium bicarbonate solution are more stable compared to the traditionally used solutions, making them more suited for the analysis of macromolecular complexes (Lemaire et al, 2001).…”

Section: Electrospray Ionization Of Biomacromoleculesmentioning

confidence: 99%

Investigation of intact protein complexes by mass spectrometry

Heck

Heuvel

2004

Mass Spectrometry Reviews

553

554

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I. Introduction 00 II. Electrospray Ionization of Biomacromolecules 00 III. Mass Analyzers for Biomacromolecular Mass Spectrometry 00 A. Collisional Cooling and/or Focusing 00 B. Alternative Mass Analyzers 00 IV. Solution‐Phase Properties of Proteins Complexes 00 A. Protein–Protein Interactions 00 B. Cooperativity and Cofactors 00 C. Dynamics of Assembly 00 V. Gas‐Phase Properties of Protein Complexes 00 A. Tandem Mass Spectrometry 00 B. Charge Separation in Protein Dissociation 00 VI. Future Outlook 00 Acknowledgments 00 References 00 Mass spectrometry has grown in recent years to a well‐accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non‐covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non‐covalent protein–protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights. © 2004 Wiley Periodicals, Inc., Mass Spec Rev

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Stabilization of Gas-Phase Noncovalent Macromolecular Complexes in Electrospray Mass Spectrometry Using Aqueous Triethylammonium Bicarbonate Buffer

Cited by 79 publications

References 29 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

Mass spectrometric determination of association constants of adenylate kinase with two noncovalent inhibitors

Investigation of intact protein complexes by mass spectrometry

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