title: Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry.
abstract: Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system. Graphical Abstract ᅟ.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N. Pine Street, Room 723, Baltimore, MD, 21201, USA.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N. Pine Street, Room 723, Baltimore, MD, 21201, USA.
authors.forename: Jianshi
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N. Pine Street, Room 723, Baltimore, MD, 21201, USA. mkane@rx.umaryland.edu.
authors.forename: Maureen A
authors.initials: MA
authors.lastname: Kane
mesh_terms: D000465:Algorithms; D000818:Animals; D007477:Ions / Q000032:analysis; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D011994:Recombinant Proteins / Q000032:analysis; D054840:Retinol-Binding Proteins, Cellular / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: C581954:CrbpI protein, mouse; D007477:Ions; D011994:Recombinant Proteins; D054840:Retinol-Binding Proteins, Cellular
keywords: Cellular retinol-binding protein; Native mass spectrometry; Noncovalent protein complex; Quantification; Vitamin A