Gas Phase Stabilization of Noncovalent Protein Complexes Formed by Electrospray Ionization

El-Hawiet

Schnier

et al. 2012

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222

321

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein-ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Section: In-source Dissociationmentioning

confidence: 99%

Section: In-source Dissociationmentioning

confidence: 99%

Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

El-Hawiet

Schnier

et al. 2012

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222

321

“…In extreme cases, in-source dissociation results in false negatives, wherein no ligand-bound protein ions are detected by ESI-MS [15]. However, using gentle sampling conditions, along with stabilizing solution or gas phase additives, it is sometimes possible to preserve the labile protein-ligand complexes during ESI-MS analysis and to quantify their association constants [17,18].…”

Section: Introductionmentioning

confidence: 99%

Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

Liu

Klassen

2011

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104

The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH 3 (CH 2 ) x COOH, where x=6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants (K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize insource dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.

“…In the extreme case, where no PL ions survive, in-source dissociation will result in a false negative. Recently, it was shown that solution or gas-phase additives can, in some instances, protect complexes from in-source dissociation [12,15]. However, this approach does have its limitations and the detection of very labile gas-phase complexes, which rapidly dissociate at ambient temperature, by ES-MS remains problematic.…”

mentioning

confidence: 99%

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Liu

et al. 2010

Self Cite

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying proteinligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L ref ), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein-ligand complex in the gas phase, is added to the solution. The fraction of P bound to L ref , which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. The assay is based on the direct detection and quantification of the abundance (Ab) of ligand-bound and unbound protein ions in the gas phase, e.g., PL nϩ and P nϩ , respectively. A key assumption is that the measured abundance ratio (R) is equivalent to the equilibrium concentration ratio of ligand-bound and free protein in solution, eq 1: The direct ES-MS assay has been used to measure affinities for a range of protein-ligand complexes, including antibody-antigen, lectin-carbohydrate, enzymesubstrate/inhibitor complexes and, in many instances, the K a values agree well with constants obtained by other analytical methods, including isothermal titration calorimetry (ITC), surface plasmon resonance, and frontal affinity chromatography MS [4 -10]. However, there have also been reports of protein-ligand complexes that could not be detected by ES-MS or, if detected, the relative abundance of ligand-bound and unbound protein ions did not match the distribution expected in solution, with less binding observed in the gas phase [11][12][13][14]. These anomalous results are often due the occurrence of in-source dissociation, whereby the gaseous complexes undergo partial or complete dissociation during ES-MS analysis. If the gas-phase PL ions are kinetically labile and undergo dissociation during analysis, the magnitude of the measured R value and, correspondingly, the K a value will be artificially low. In the extreme case, where no PL ions survive, in-source dissociation will result in a false negative. Recently, it was shown that solution or gas-phase additives can, in some instances, protect complexes from in-source dissociation [12,15]. However, this approach does have its limitations and the detection of very labile gas-phase complexes, which rapidly dissociate at ambient temperature, by ES-MS remains problematic. Here, we describe an indirect ES-MS approach to quantify protein-ligand interactions that are highly Address reprint requests to Dr.