Non-covalent binding of endogenous ligands to recombinant cellular retinol-binding proteins studied by mass spectrometric techniques

Recent developments in mass spectrometry have demonstrated the capability o f this technique to transfer non-covalent protein complexes, involving low and high molecular weight ligands, from a condensed state to the gas phase. In this work, electrospray mass spectrometry with a quadrupole analyzer (E S-MS) and matrix-assisted laser desorption/ionization time-of-flight mass s pectrometry (MALDI-TOFMS) were used to analyze the non-covalent association between recombinant rat cellular retinol-binding protein type-I (CRBP) wit h its specific ligand, all-trans retinol (vitamin A), and with fatty acids. Under denaturing conditions, MALDI-TOFMS and ES-MS techniques allowed dete rmination of the molecular weight of apo-CRBP with good accuracy (<0.01%) a nd to identify a protein fraction (similar to 20%) retaining the initial me thionine. By adding saturating amounts of vitamin A, ES-MS studies on the p rotein in the holo-form under native conditions allowed detection of retino l bound within the cavity together with water molecules, as expected from i ts crystal structure. ES mass spectra of CRBP in the native state were also recorded under non-denaturing conditions, with the aim to study non-covale nt interactions between CRBP and non-specific ligands such as fatty acids, bound to the protein as a result of expression in various strains of E. col i grown in different media. Since ES mass spectra do not elucidate which sp ecies interact with the protein, in order to investigate the ligands possib ly retained in the active site of recombinant CRBP, liquid chromatography/E S-tandem mass spectrometry was used. In particular, this technique was appl ied to identify and quantify fatty acids bound to CRBP. Quantitative data i ndicated the presence of a few fatty acids at a total concentration lower t han 2% of that of the protein. Similar findings were observed for the homol og rat cellular retinol-binding protein type-II, demonstrating the high deg ree of purity and homogeneity of apo-CRBP preparations derived from gene ex pression. Copyright (C) 2001 John Wiley & Sons, Ltd.