Bioassay-directed fractionation for discovery of bioactive neutral lipids guided by relative mass defect filtering and multiplexed collision-induced dissociation

Abstract

We report a synergistic method using bioassay-directed liquid chromatography fractionation and time-of-flight mass spectrometry to guide and accelerate bioactive compound discovery. To steer purification and assays toward anticipated neutral lipid activators of a constitutive androstane receptor splice variant, a relative mass defect filter was calculated, based on the ratio of the mass defect to the measured ion mass, and used to reduce the number of candidate ion masses. Mass measurements often lack sufficient accuracy to provide unambiguous assignments of elemental compositions, and since the relative mass defect reflects fractional hydrogen content of ions, this value is largely determined by the hydrogen content of a compound's biosynthetic precursors. A relative mass defect window ranging from 600-1000 ppm, consistent with an assortment of lipids, was chosen to assess the number of candidate ions in fractions of fetal bovine serum. This filter reduced the number of candidate ion m/z values from 1345 to 892, which was further reduced to 21 by intensity and isotope filtering. Accurate mass measurements from time-of-flight mass spectrometry and fragment ion masses generated using nonselective collision-induced dissociation suggested dioctyl phthalate as one of few neutral lipid constituents in the active fraction. The identity of this compound was determined to be di(2-ethylhexyl) phthalate using GC/MS, and it was ranked as a promising candidate for reporter assay screening.

Figure 1

Work flow for discovery and identification of bioactive compounds.

Figure 2

(A) Total ion chromatogram from the LC separation of the active fraction from Process 1 (fraction 17), highlighting the retention time window selected for summation of mass spectra. (B) Histogram representing the frequency of relative mass defects for the 1345 discrete m/z values from summing all mass spectra from retention times 4–8 min. Highlighted in orange is the range of RMD values of interest, 600–1000 ppm, targeting likely neutral lipids. (C) Two summed spectra obtained from the m/z values in the selected range in (B). The top spectrum presents all m/z values from this range while the bottom spectrum presents the 21 remaining m/z values after intensity filtering and deisotoping. Peak A = m/z 214, B = m/z 251, C = m/z 279, D = m/z 374, E = m/z 391, F = m/z 448, G = m/z 638, H = m/z 680. (D) Extracted ion chromatograms depicting the second fractionation scheme (Process 2) in which the three ion m/z values of interest are distributed among two collected fractions.

Figure 3

The relationship between relative mass defect and % hydrogen for an assortment of organic compounds. Selected for by the dashed line is the range of interest, from 600 to 1000 ppm.

Figure 4

Structures of three isomers of dioctyl phthalate: (DEHP) di(2-ethylhexyl) phthalate, (DIOP) diisooctyl phthalate, and (DNOP) di-n-octyl phthalate.

Figure 5

GC/MS extracted ion chromatograms for m/z 149 for phthalate ester standards and extract of FBS. The peaks eluting at 12.22 and 13.92 min correspond to DEHP and DNOP, respectively, confirming the presence of DEHP in extracts of FBS. (A) 5 µM DEHP and 5 µM DNOP standards, (B) extract of 100 µL of FBS.