New reagents for increasing ESI multiple charging of proteins and protein complexes

Self Cite

101

A Fourier-transform ion cyclotron resonance (FT-ICR) top-down mass spectrometry strategy for determining the adenosine triphosphate (ATP)-binding site on chicken adenylate kinase is described. Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), but the ability to detect protein-ligand complexes depends on their stability in the gas phase. Previously, we showed that collisionally activated dissociation (CAD) of protein-nucleotide triphosphate complexes yield products from the dissociation of a covalent phosphate bond of the nucleotide with subsequent release of the nucleotide monophosphate (Yin, S. et al., J. Am. Soc. Mass Spectrom. 2008, 19, 1199 -1208. The intrinsic stability of electrostatic interactions in the gas phase allows the diphosphate group to remain noncovalently bound to the protein. This feature is exploited to yield positional information on the site of ATP-binding on adenylate kinase. CAD and electron capture dissociation (ECD) of the adenylate kinase-ATP complex generate product ions bearing monoand diphosphate groups from regions previously suggested as the ATP-binding pocket by NMR and crystallographic techniques. Top-down MS may be a viable tool to determine the ATP-binding sites on protein kinases and identify previously unknown protein kinases in a functional proteomics study. (J Am Soc Mass Spectrom 2010, 21, 899 -907) © 2010 American Society for Mass Spectrometry P roteins function in biology through direct interaction with other molecules. A ligand can be a molecule, an atom, or an ion that binds to a specific site on the protein by a variety of means, including hydrogen bonding, Van der Waals interactions, and ionic forces. Determining the presence of protein-ligand interactions and the structures of proteinligand complexes are of critical importance in biology, and this knowledge is often essential for efforts to establish new molecular drug targets and to develop more potent medicines.A number of biophysical tools are available to characterize the interaction between a protein and its ligand(s). However, mass spectrometry (MS) offers potential advantages in sensitivity, specificity, and speed, especially compared with high-resolution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. With electrospray ionization (ESI) to ionize macromolecules without disrupting covalent bonds while maintaining weak noncovalent interactions, the ESI-MS molecular mass measurement provides direct evidence for protein-ligand associations. The proteinligand interactions are often sufficiently retained upon the transition from solution to the gas phase that the complex size and binding stoichiometry can be measured [1][2][3].Although binding stoichiometry and, in many examples, the relative and absolute solution binding affinities can be measured by ESI-MS methodologies, determining the precise ligand binding site on a target protein is not easily tractable using MS directly. Tandem mass spectrometry (MS/MS) is widely applied...

“…ESI mass spectra for nucleotide-bound AK have been reported previously [33][34][35][36] Figure 1b). This behavior is similar to our previous data for CMP-bound RNase A [10].…”

Section: Cad-ms/ms Of Ak Ak-amp and Ak-atpmentioning

confidence: 56%

Elucidating the site of protein-ATP binding by top-down mass spectrometry

Yin

Loo

2010

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101

“…These experiments take the form of 'supercharging' or 'charge reduction' depending on the solution additives. Considering first the evidence for folded structure under supercharging conditions, addition of m-NBA or sulfolane [22,23] is used to produce higher charge states than normally accessible by globular proteins and their complexes. An increase in average charge state is reported, from 10+ to 15+, for the 29 kDa carbonic anhydrase-zinc complex.…”

Section: Can Charge States Be Manipulated Without Affecting Folded Prmentioning

confidence: 99%

Do Charge State Signatures Guarantee Protein Conformations?

Hall

Robinson

2012

157

170

The extent to which proteins in the gas phase retain their condensed-phase structure is a hotly debated issue. Closely related to this is the degree to which the observed charge state reflects protein conformation. Evidence from electron capture dissociation, hydrogen/deuterium exchange, ion mobility, and molecular dynamics shows clearly that there is often a strong correlation between the degree of folding and charge state, with the most compact conformations observed for the lowest charge states. In this article, we address recent controversies surrounding the relationship between charge states and folding, focussing also on the manipulation of charge in solution and its effect on conformation. 'Supercharging' reagents that have been used to effect change in charge state can promote unfolding in the electrospray droplet. However for several protein complexes, supercharging does not appear to perturb the structure in that unfolding is not detected. Consequently, a higher charge state does not necessarily imply unfolding. Whilst the effect of charge manipulation on conformation remains controversial, there is strong evidence that a folded, compact state of a protein can survive in the gas phase, at least on a millisecond timescale. The exact nature of the side-chain packing and secondary structural elements in these compact states, however, remains elusive and prompts further research.

“…This hypothesis would imply that the charge of the protein depends on solvent surface tension following the Rayleigh equation [12]. Such a dependence could not be reproduced by experiments on either folded or unfolded proteins [1,2,[12][13][14][15], although solvent surface tension certainly plays an important role in the ESI process [1,2], and has also been suggested to explain some effects of supercharging agents [11,16].…”

Section: Introductionmentioning

confidence: 99%

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

Marchese

Grandori

Carloni

et al. 2012

Identifying the key factor(s) governing the overall protein charge is crucial for the interpretation of electrospray-ionization mass spectrometry data. Current hypotheses invoke different principles for folded and unfolded proteins. Here, first we investigate the gas-phase structure and energetics of several proteins of variable size and different folds. The conformer and protomer space of these proteins ions is explored exhaustively by hybrid Monte-Carlo/molecular dynamics calculations, allowing for zwitterionic states. From these calculations, the apparent gas-phase basicity of desolvated protein ions turns out to be the unifying trait dictating protein ionization by electrospray. Next, we develop a simple, general, adjustable-parameter-free model for the potential energy function of proteins. The model is capable to predict with remarkable accuracy the experimental charge of folded proteins and its well-known correlation with the square root of protein mass.