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An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data. 

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance.