Structural Elucidation and Relative Quantification of Fatty Acid Double Bond Positional Isomers in Biological Tissues Enabled by Gas-Phase Charge Inversion Ion/Ion Reactions

Abstract

Fatty acids (FAs) contain a vast amount of structural diversity, and differences in fatty acid structure have been associated with various disease states. Accurate identification and characterization of fatty acids is critical to fully understand the biochemical roles these compounds play in disease progression. Conventional tandem mass spectrometry (MS/MS) workflows do not provide sufficient structural information, necessitating alternative dissociation methods. Gas-phase charge inversion ion/ion reactions can be used to alter the ion type subjected to activation to provide improved or complementary structural information. Herein, we have used an ion/ion reaction between fatty acid (FA) anions and magnesium tris-phenanthroline [Mg(Phen)₃] dications to promote charge remote fragmentation of carbon-carbon bonds along the fatty acid chain, allowing for localization of carbon-carbon double bond (C=C) positions to successfully differentiate monounsaturated fatty acid isomers. Relative quantification was also performed to obtain the relative abundance of fatty acid isomers in different biological tissues. For example, the relative abundance of FA 18:1 (9) was determined to vary across regions of rat brain, rat kidney, and mouse pancreas, and FA 16:1 (9) was found to have a higher relative abundance in the dermis layer compared to the sebaceous glands in human skin tissue.

Keywords: double bond; fatty acids; imaging mass spectrometry; ion/ion reactions; positional isomers.

Scheme 1.

A gas phase charge inversion ion/ion reaction between a singly charged FA anion and a doubly charged [Mg(Phen)₃]²⁺ reagent cation results in the formation of a [FA − H + Mg(Phen)₂]⁺ complex ion, where one phenanthroline is lost and the fatty acid anion forms a complex with magnesium phenanthroline. Activation of this complex results in the loss of an additional phenanthroline and fragmentation along the fatty acyl chain.

Figure 1.

Gas-phase charge inversion ion/ion reactions performed using a FA 18:1 (9Z) synthetic standard and Mg(Phen)₃ involve (a) isolation of FA precursor anions generated via MALDI, (b) isolation of Mg(Phen)₃ reagent dications generated via ESI, (c) ion/ion reaction of FA 18:1 (9Z) and Mg(Phen)₃ to produce a charge inverted [FA – H + Mg(Phen)₂]⁺ singly charged complex product ion, and (d) SORI CID for the complex product ion to produce [FA – H + MgPhen]⁺ resulting from the loss of one phenanthroline ligand as well FA charge remote fragmentation. The spectral inset shows that charge remote fragmentation occurs, except at the the double bond, where the “dip” in fragment ion intensity along the aliphatic FA chain is highlighted in orange and indicates the position of the double bond. Note: squares indicate positive ion mode analysis, circles indicate negative mode analysis, filled shapes represent ion isolation events, and hollow shapes represent full scan acquisition. Asterisks denote harmonics/electronic noise. All spectra are single scan measurements, except for the SORI CID spectrum (8 average scans).

Figure 2.

Gas-phase charge inversion ion/ion reactions performed using fatty acid synthetic standards and Mg(Phen)₃ involve (a) SORI CID of the FA 18:1 (9Z) complex product ion and (b) SORI CID of the FA 18:1 (11Z) complex product ion, which both result in the loss of one phenanthroline ligand as well FA charge remote fragmentation. The spectral insets show that charge remote fragmentation occurs until the double bond, where the “dip” in fragment ion intensity along the aliphatic FA chain is highlighted in color and indicates the position of the double bond. Note: squares indicate positive ion mode analysis, circles indicate negative mode analysis, filled shapes represent ion isolation events, and hollow shapes represent full scan acquisition. Asterisks denote harmonics/electronic noise. All spectra are 8 average scan measurements.

Figure 3.

Gas-phase charge inversion ion/ion reactions performed using a fatty acid synthetic standards and Mg(Phen)₃ involve (a) SORI CID for the FA 16:1 (9Z) complex product ion and (b) SORI CID for the FA 16:1 (6Z) complex product ion, which both result in the loss of one phenanthroline ligand as well FA charge remote fragmentation. The spectral insets show that charge remote fragmentation occurs until the double bond, where the “dip” in fragment ion intensity along the aliphatic FA chain is highlighted in color and indicates the position of the double bond. Note: squares indicate positive ion mode analysis, circles indicate negative mode analysis, filled shapes represent ion isolation events, and hollow shapes represent full scan acquisition. Asterisks denote harmonics/electronic noise. All spectra are 8 average scan measurements.

Figure 4:

(a) Ion image for m/z 281.249 in rat brain, tentatively identified as FA 18:1 by an accurate mass measurement (5.3 ppm). (b) SORI CID of the [FA − H + Mg(Phen)₂]⁺ complex product ion produces [FA – H + MgPhen]⁺ resulting from the loss of one phenanthroline ligand as well as FA charge remote fragmentation. Both i’ and k fragment ions in the spectrum and the intensity ratio of the b+· and d’ fragment ions (highlighted in brown) indicate the presence FA 18:1 (9) and FA 18:1 (11) isomers in the tissue. Note: squares indicate positive ion mode analysis, circles indicate negative mode analysis, filled shapes represent ion isolation events, and hollow shapes represent full scan acquisition. Asterisks denote harmonics/electronic noise. Spectrum is 2 average scan measurements.

Figure 5.

(a) Mixtures of FA 18:1 (9Z) and FA 18:1 (11Z) fatty acid standards are used to calibrate the d’/(d’+b+·) fragment ion intensity ratio obtained via SORI CID of the [FA – H + Mg(Phen)₂]⁺ complex product ion . Each measurement represents the average of 6 spectra and each spectrum is 3 average scan measurements. (b) The ion/ion reaction coupled with SORI CID reveals differences in the relative content of FA 18:1 (9) and FA 18:1 (11) in six different regions of rat brain tissue. Each measurement is the average of 5 spectra and each spectrum represents 3 average scan measurements (c) H&E stained tissue showing regions of interest

Figure 6.

(a) Mixtures of FA 18:1 (9Z) and FA 18:1 (11Z) fatty acid standards are used to calibrate the d’/(d’+b+·) fragment ion intensity ratio obtained via SORI CID of the [FA – H + Mg(Phen)₂]⁺ complex product ion . Each measurement is the average of 6 spectra and each spectrum represents 3 average scan measurements (b) The ion/ion reaction coupled with SORI CID reveals differences in the relative content of FA 18:1 (9) and FA 18:1 (11) in two different regions of rat kidney tissue and in mouse exocrine pancreas tissue. Each measurement is the average of 5 spectra and each spectrum represents 3 average scan measurements (c) H&E-stained tissue showing regions of interest. Note: this mouse pancreas is largely comprised of exocrine pancreas.

Figure 7.

(a) Mixtures of FA 16:1 (9Z) and FA 16:1 (6Z) fatty acid standards are used to calibrate the c’/(c’+b+·) fragment ion intensity ratio obtained via SORI CID of the [FA – H + Mg(Phen)₂]⁺ complex product ion . Each measurement is the average of 6 spectra and each spectrum represents 3 average scan measurements (b) The ion/ion reaction coupled with SORI CID reveals differences in the relative content of FA 16:1 (9) and FA 16:1 (6Z) in two different regions of human skin tissue. Each measurement represents the average of 5 spectra and each spectrum is 3 average scan measurements (c) H&E-stained tissue showing regions of interest.