Method for Distinguishing Specific from Nonspecific Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry

Sun, Jiangxiao; Kitova, Elena N.; Wang, Weijie; Klassen, John S.

doi:10.1021/ac0522005

Cited by 161 publications

(261 citation statements)

References 26 publications

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“…A reference protein (Lyz) was also added to monitor for the occurrence of non-specific ligand binding to scFv during the ES process [19]. At these concentrations, 60% of the scFv is expected to be bound to 1 in solution.…”

Section: Scfv-monosaccharide Bindingmentioning

confidence: 99%

“…In contrast, ES-MS analysis of a solution of scFv (10 M) and 4 (59 M) clearly identified the presence of gas-phase ions corresponding to the (scFv ϩ 4) complex, Figure 2b. The K a value determined directly from the ES mass spectrum, following correction for nonspecific ligand binding [19], was (1.6 Ϯ 0.5) ϫ 10 5 M Ϫ1 . Shown in Figure 2c is a representative ES mass spectrum acquired for a solution of scFv (10 M), 1 (1 mM) and 4 (59 M).…”

Section: Scfv-monosaccharide Bindingmentioning

confidence: 99%

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Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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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.

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Section: Scfv-monosaccharide Bindingmentioning

confidence: 99%

Section: Scfv-monosaccharide Bindingmentioning

confidence: 99%

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

El-Hawiet

Kitova

Liu

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…We further investigated the interaction between recombinant bundlin and LacNAc by the nanoelectrospray mass spectrometry (nanoES-MS) (Sun et al, 2006) technique. This ultra-sensitive assay has been shown to reveal affinities for protein-glycan complexes that agree with values obtained by other assays including isothermal titration (Sun et al, 2006).…”

Section: Bundlin Is a Lacnac-specific Lectinmentioning

confidence: 99%

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

et al. 2007

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SummarySynthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAcspecific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K assoc), to synthetic LacNAc-Benzene and LacNAc-O(CH2)8CONH2 glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express a bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the a bundlin monomer. Collectively, these results suggest that a bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of a bundlin-expressing EPEC strains to host intestinal epithelial cells.

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“…Nanospray [34 -36] is often a gentle ionization method of allowing the use of less harsh conditions including a lower heated capillary temperature, the absence of heater gas, and a lower spray voltage while also providing high ionization efficiency for small-sample consumption. These advantages favor the application of nanospray to study protein conformation and noncovalent complexes [37][38][39].…”

mentioning

confidence: 99%

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Guo

Zhang

Song

et al. 2009

J. Am. Soc. Mass Spectrom.

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Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to Ϫ80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution. C haracterization of native conformations and noncovalent complexes of proteins is of great significance to understanding a variety of biological processes at the molecular level. Electrospray ionization mass spectrometry (ESI-MS) [1, 2] has grown into a powerful technique in this field. Besides its capability of handling large biomolecules, the advantages of ESI-MS over other spectroscopic and biophysical methods include high analysis speed, minimal sample consumption, and the characterization of individual conformational states that may coexist in solution at equilibrium [3][4][5]. The charge-state distribution (CSD) in the ESI mass spectra represents a specific conformational state of protein [6 -9]. Broad CSDs at high charge states are generally associated with unfolded proteins, whereas narrower distributions centered on lower charge states are treated as characteristics of folded proteins [6, 10 -15]. However, the harsh conditions during the ionization process in MS are often detrimental to the preservation of protein conformation and the survival of noncovalent complexes. Many studies have examined the influence of the ionization process and the operating settings of ESI, including the curtain gas [16,17], the pressure in the ion source [18 -20], the temperature [21][22][23][24][25][26],...

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Method for Distinguishing Specific from Nonspecific Protein−Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry

Cited by 161 publications

References 26 publications

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

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