Electrospray mass spectrometry for the direct accurate mass measurement of ligands in complex with the retinoid X receptor α ligand binding domain

Abstract

Accurate mass measurements are often used in the structural determination of unknown compounds of low molecular mass (i.e., below ϳ500 Da). Recently, it has been shown that accurate mass measurements also can be made on small denatured proteins (i.e., M r , ϳ17,000) to confirm their amino acid composition and identify the presence of isoforms. In the current report, we present nondenaturing electrospray (ES) mass spectrometry data on the direct accurate mass measurement of ligands in complex with the retinoid X receptor ligand binding domain (RXR LBD; M r 31,370.92). Average mass errors were below 0.198 Da, 6.3 ppm (standard deviation [SD], 0.146; n ϭ 10) for low-affinity fatty acid agonists analyzed in complex with the RXR LBD. Protein consumption was less than 15 pmol, with fatty acid ligands present at concentrations corresponding to their median effective concentration value (low micromolar, determined in transfection assays). Although determination of fatty acid mass was only sufficiently accurate to give nominal mass values, measurements were of sufficient accuracy to assign fatty acid chain length, degree of unsaturation, or cyclization. Using 17␤-estradiol as a control, the ability to observe specific ligand binding is shown for both high-and low-affinity RXR␣ agonists. In addition, binding of a novel synthetic receptor agonist XCT0315908 to the RXR␣ LBD is reported. This compound showed a high degree of complex formation, and the receptor-ligand complex could be mass measured with an average mass error of Ϫ0.024 Da, 0.8 ppm (SD, 0.092; n ϭ 9). Thus, specific binding of both nanomolar and micromolar affinity ligands to a nuclear receptor LBD can be directly observed using nondenaturing ES mass spectrometry and accurate mass measurements additionally can be made on intact complexes in the same experiment. This methodology also is applicable when ligands are present as components of mixtures. (J Am Soc Mass Spectrom 2005, 16, 1631-1640

Structural and Functional Insights into the Ligand-binding Domain of a Nonduplicated Retinoid X Nuclear Receptor from the Invertebrate Chordate Amphioxus

“…3E). The fact that 9-cis-RA presents a higher affinity than DHA for HsRXR␣ is in agreement with the observations of Lengqvist et al (48,50). Affinity rankings established by titration experiments for both RXR homologues were further confirmed by direct in solution competition experiments involving a mixture of equimolar amounts of ligands (10 M each) and 10 M RXR (data not shown).…”

Novel 1α,25-Dihydroxyvitamin D3Analogues with the Side Chain at C12

“…Changes in electrostatic and H-bond contacts upon chemical modification of the ligand are directly reflected in the ESI-MS data, which allows a qualitative classification of ligands. This qualitative classification obtained by ESI-MS is similar to that obtained by classical competition experiments. ,− ESI-MS titration experiments were performed for hVDRΔ LBD binding with 2a , 2b , 2c , 1α,25(OH) 2 D 3 , and as negative control 9- cis -retinoic acid, ligand for the retinoid NRs with no known affinity for VDR. The dominant species were found to be [M + 12H] 12+ and [M + 11H] 11+ charged states of the monomeric hVDRΔ LBD.…”

“…Biological Activity. The strength of the interaction of 2a , 2b , and 2c for hVDRΔ LBD, was evaluated by titration and competition experiments with electrospray ionization mass spectrometry (ESI-MS), a technique that allows characterization of noncovalent interactions and is accordingly increasingly being used to study ligand binding. − This methodology is used to evaluate electrostatic and H-bond specific contacts involved in the binding of a ligand to a receptor. Changes in electrostatic and H-bond contacts upon chemical modification of the ligand are directly reflected in the ESI-MS data, which allows a qualitative classification of ligands.…”

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

“…They are assigned to the gluconoylation (+C 6 H 10 O 6 , MW 178.14) and phosphogluconoylation (+C 6 H 11 O 9 P, MW 258.12), respectively, of the His 6 -affinity tag (Geoghegan et al 1999). Such posttranslational modifications were previously observed for nuclear receptor proteins overexpressed in Escherichia coli (Lengqvist et al 2002(Lengqvist et al , 2004(Lengqvist et al , 2005Stehlin-Gaon et al 2003). The deconvoluted molecular mass of 43,814 Da did not agree with the molecular mass based on the known amino acid sequence (43,947 Da).…”

“…Several reports have shown the potential of ESI-MS to characterize the binding of NR ligands to their receptor in terms of stochiometry, specificity, and stability (Greschik et al 2002;Lengqvist et al 2002Lengqvist et al , 2004Lengqvist et al , 2005Bitsch et al 2003;Potier et al 2003;Stehlin-Gaon et al 2003;Sanglier et al 2004). ESI-MS has been successfully used to assess noncovalent complex stability in the vacuum of the mass spectrometer by analyzing their resistance to dissociation Wu et al 1997;van der Kerk-van Hoof and Heck 1999) and for measuring solution binding constants (Jorgensen et al 1998;Sannes-Lowery et al 2000;Daniel et al 2003;Gabelica et al 2003;Wendt et al 2003;Tjernberg et al 2004;Wortmann et al 2005;De Vriendt et al 2006).…”