Photochemical Tagging for Quantitation of Unsaturated Fatty Acids by Mass Spectrometry

Abstract

Fatty acid (FA) profiling provides phenotypic information and is increasingly used in a broad range of biological and biomedical studies. Quantitation of unsaturated FAs with confident carbon-carbon double bond (C═C) location assignment is both sample and time consuming using traditional gas chromatography mass spectrometry analysis. In this study, we developed a rapid, sensitive, and quantitative method for profiling unsaturated FAs without using chromatographic separations. This method was based on a combination of in-solution photochemical tagging of a C═C in FAs and a subsequent gas-phase detagging via tandem (neutral loss scan) mass spectrometry. It enabled quantitation of unsaturated FAs from various biological samples (blood, plasma, and cell lines). More importantly, quantitative information on FA C═C location isomers, which was traditionally overlooked, could now be obtained and applied to studying FA changes between normal and cancerous human prostate cells.

Figure 1

Negative ion mode nanoESI mass spectra of an equal molar mixture of FA 17:1(10Z) and FA 18:1(9Z) (3.5 µM each in acetone/H₂O =50/50, v/v with 0.1% NH₄OH added): (a) before and (b) after photochemical tagging by acetone. (c) MS² beam-type CID (35 eV) of tagged FA 17:1 (m/z 325). Negative ion mode nanoESI-MS of FA extract from human plasma: (d) before and (e) after acetone tagging. FA 17:1(10Z) was used as the internal standard (IS, 7.5 µM). (f) 58 Da neutral loss scan (NLS) after acetone tagging. The number of “*” sign in FA annotation stands for the number of acetone molecules tagged onto a FA.

Figure 2

(a) Extracted ion chromatogram of intact and acetone-tagged FA 17:1 and FA 18:1 during photochemical reaction. (b) NLS (58 Da) of tagged FA 17:1 and FA 18:1. Inset shows the calibration curve for FA 18:1(9Z) based on NLS (IS: FA 17:1). Photochemical reaction MS spectrum of FA 20:4 (5Z, 8Z, 11Z, 14Z) using different reaction solvent conditions: (a) acetone/water (50/50, v/v) and (b) ethanol/acetone/water (40/30/30, v/v/v).

Figure 3

Analysis of FA C=C location isomers from human plasma. (a) PB-MS/MS of tagged FA 18:1 (m/z 339.3). Two isomers with C=C at T9 and T11 positions are detected. (b) Calibration curve for relative quantitation of FA 18:1 T9 and T11 isomers. (c) Calibration curve for total FA 18:1 quantitation based on 58 Da NLS of FA 18:1 mixtures consisting of 91.5% T9 and 8.5% T11 isomers. (d) PB-MS/MS of an equal molar mixture of ω-3 and ω-6 FA 18:3 isomers (m/z 343.3 lithiated ions). (e) PB-MS/MS of zoomed-in regions for C=C diagnostic ions of FA 18:3 from human plasma. ω-3 FA 18:3 is more abundant than ω-6 isomer.

Figure 4

(a) Quantitation of unsaturated FAs in human plasma. (b) Comparison of the amounts of major unsaturated FAs in RWPE1 and PC3 cells. Inset shows %composition of Δ9 and Δ11 isomers of FA 18:1 in RWPE1 and PC3 cells. Error bars represent standard deviation, n = 3. Differences between the FAs in RWPE1 cells and PC3 cells were evaluated for statistical significance using the two-tailed Student’s t-tests (***p<0.0005).

SCHEME 1

Selective detection of unsaturated FAs from complex mixtures using in-solution acetone tagging (PB reaction) followed by gas-phase de-tagging in MS/MS.