Detection of nucleic acid-nuclear hormone receptor complexes with mass spectrometry

Abstract

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR·RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR·RXR·DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR. (J Am Soc Mass Spectrom 2010, 21, 635-645)

The retinoid X receptors and their ligands

“…Both non-denaturing nanoelectrospray ionization (nano-ESI) and high-mass matrix-assisted laser desorption ionization (MALDI) mass spectrometric methods were used to demonstrate that the RXR–RAR heterodimer bound to a DR-5 RARE in the presence of 9-cis-RA and that RXR was upstream (5′), in contrast to the DR-1 RARE in which RAR was upstream [6]. Crosslinking was used to stablilize the complex for MALDI, but did not necessarily stabilize the bound ligand.…”

Analytical Approaches for Assessing Aggregation of Protein Biopharmaceuticals

“…In particular, mass spectrometry-based techniques are under development to determine higher-order structure and conformational dynamics of bioproducts [58,59]. This methodological approach has been largely proved as powerful and highly resolutive for the monitoring and screening of protein aggregation and formation of protein complexes in different biological contexts [60][61][62] and it has been even applied to determine the formation of protein oligomers in the basic research of fine protein-protein [63] and protein-nucleic acid [64] interactions. Intriguingly, mass spectrometry is used to analyze inhibitors of protein aggregation in the development of antiAlzheimer's disease drugs [65].…”

A New, Modular Mass Calibrant for High-Mass MALDI-MS

“…[14][15][16] High accuracy mass information is key, for example, for determining the stoi-chiometry of protein-protein, 3,4,17,18 protein-DNA, 19 or protein-RNA complexes. 20 Exact mass information is also used for the determination of the degree and the kind of post-translational modifications (PTMs), 7 PEGylations, 21 as well as for determining truncation of the primary sequence.…”