Multidimensional mass spectrometry-based shotgun lipidomics analysis of vinyl ether diglycerides

Abstract

Diglycerides play a central role in lipid metabolism and signaling in mammalian cells. Although diacylglycerol molecular species comprise the majority of cellular diglycerides that are commonly measured using a variety of approaches, identification of extremely low abundance vinyl ether diglycerides has remained challenging. In this work, representative molecular species from the three diglyceride subclasses (diacyl, vinyl ether, and alkyl ether diglycerides; hereafter referred to as diradylglycerols) were interrogated by mass spectrometric analysis. Product ion mass spectra of the synthesized diradylglycerols with varied chain lengths and degrees of unsaturation demonstrated diagnostic fragmentation patterns indicative of each subclass. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis of mouse brain and heart lipid extracts were performed using the identified informative signature product ions. Through an array of tandem mass spectrometric analyses utilizing the orthogonal characteristics of neutral loss scanning and precursor ion scanning, the differential fragmentation of each subclass was exploited for high-yield structural analyses. Although molecular ion mass spectra readily identified diacylglycerol molecular species directly from the hexane fractions of tissue extracts enriched in nonpolar lipids, molecular ion peaks corresponding to ether-linked diglycerides were not observable. The power of MDMS-SL utilizing the tandem mass spectrometric array analysis was demonstrated by identification and profiling of individual molecular species of vinyl ether diglycerides in mouse brain and heart from their undetectable molecular ion peaks during MS(1) analysis. Collectively, this technology enabled the identification and profiling of previously inaccessible vinyl ether diglyceride molecular species in mammalian tissues directly from extracts of biologic tissues.

Figure 1

Tandem mass spectra of product ions of NH4⁺ adducts of synthetic diacyl, vinyl ether, alkyl ether diglycerides. The tandem mass spectra are presented for diacyl (D), vinyl ether or plasmenyl (p) and alkyl ether (a) 16:0–18:1 diglycerides (left panel) and for 18:0–20:4 diglycerides (right panel). The fragment ions at m/z 339 (left panel) and at m/z 361 (right panel) correspond to the cationic dehydrated 18:1 and 20:4-monoacylglycerol product ions, respectively, which are generated from the neutral loss of NH₃ and corresponding sn-1 moieties (16:0 or 18:0 fatty acid for diacylglycerols, vinyl alcohol for vinyl ether diglycerides, and alcohol for alkyl ether diglycerides). The fragment ions at m/z 240 (left panel) and at m/z 268 (right panel) are signature product ions for vinyl ether diglycerides that are absent in the spectra of diacylglycerols and alkyl ether diglycerides, which are NH₄⁺ adducts of the 16 carbon and 18 carbon terminal alkynes derived from 16:0 and 18:0 vinyl alcohols, respectively.

Figure 2

Tandem mass spectra of product ions of Li⁺ or Na⁺ adducts of synthetic diacyl, vinyl ether, and alkyl ether diglycerides. The tandem mass spectra are presented for the Li⁺ adducts of diacyl (D), vinyl ether or plasmenyl (p) and alkyl ether (a) 18:0–20:4 diglycerides (left panel) and their corresponding Na⁺ adducts (right panel). Structurally informative fragment ions are present in tandem mass spectra of the diglyceride Li⁺ adduct (e.g., the product ion at m/z 311 representing the Li⁺ adduct of 20:4 fatty acid; the product ions at m/z 347, m/z 331 and m/z 333 representing the fragment ions after neutral loss of 20:4 fatty acid from diacyl, vinyl ether, or alkyl ether diglyceride molecular ions, respectively; and the product ion at m/z 341 resulting from the neutral loss of 20:4 fatty acid lithiated salt from the diacylglycerol molecular ion). In contrast, Na⁺ adducts of vinyl and alkyl ether diglycerides do not fragment under the experimental conditions employed, while the Na⁺ adducts of diacylglycerol species do fragment but generate only low abundance structural product ions.

Figure 3

Tandem mass spectra of product ions of protonated DMG-derivatized synthetic diacyl, vinyl ether, alkyl ether diglycerides. The tandem mass spectra are presented for diacyl (D), vinyl ether or plasmenyl (p) and alkyl ether (a) 16:0–18:1 diglyceride-DMG derivatives (left panel) and for 18:0–20:4 diglyceride-DMG derivatives (right panel). Product ion scanning of both diacyl and alkyl ether diglyceride-DMG derivatives demonstrates the neutral loss of DMG as the predominant fragment ion (i.e., m/z 577 or m/z 563 for diacyl or alkyl ether 16:0–18:1 diglyceride, respectively; and at m/z 627 or m/z 613 for diacyl or alkyl ether 18:0–20:4 diglyceride, respectively). In contrast, in addition to the fragment ion at m/z 561 or m/z 611 from the neutral loss of DMG, product ion scanning of protonated vinyl ether diglyceride-DMG derivatives presents structurally informative fragment ions that include the fragment ion at m/z 279 or m/z 307 resulting from the neutral loss of DMG and 18:1 or 20:4 fatty acid, and the fragment ion at m/z 424 or m/z 446 resulting from the neutral loss of 16:0 or 18:0 vinyl alcohol, for 16:0–18:1 or 18:0–20:4 vinyl ether diglycerides, respectively.

Figure 4

Identification and profiling of vinyl ether diglyceride molecular species in mouse brain tissues. Hexane extracts of mouse brain were prepared as described in Materials and methods. Brain hexane extracts were diluted in the presence of 10 mM CH₃CO₂NH₄, and the NH₄⁺ adducts of diradylglycerols were analyzed in the positive-ion mode utilizing a full MS¹ scan and the NLS as described in Materials and methods. The top mass spectrum represents the MS¹ full scan that reveals predominantly the diacylglycerol molecular ions that are also present in the tandem mass spectrum of neutral loss (NL) of both NH₃ and H₂O. The three bottom spectra are the tandem mass spectra from neutral loss of NH₃ and a vinyl alcohol (or alternatively an isobaric odd chain length fatty acid, or an isomeric fatty alcohol which were excluded (see text)). Note that the ion peaks detected in NLS of 257, 283 or 285 amu are either undetectable or barely detectable in the full MS analysis and NLS of NH₃ and H₂O, as shown by dashed lines.

Figure 5

Identification of vinyl ether diglyceride molecular species present in mouse heart. Hexane extracts of mouse heart were prepared as described in Materials and methods. Heart hexane extracts were diluted in the presence of 10 mM CH₃CO₂NH₄, and the NH₄⁺ adducts of the diradylglycerols were analyzed in the positive-ion mode utilizing full MS¹ scan, NLS and PIS as described in Figure 4 and ESM Figures S3 and S4. The top full MS¹ scan reveals predominantly diacylglycerol molecular ions that are also present in the tandem mass spectrum of neutral loss (NL) of NH₃ and H₂O. NLS of 257 amu represents the loss of NH₃ and 16:0 vinyl alcohol. PIS of m/z 240 corresponds to the tandem mass spectrometric analysis of the NH₄⁺ adduct of the 16 carbon saturated terminal alkyne derived from 16:0 vinyl alcohol. PIS of m/z 385 corresponds to the tandem mass spectrometric analysis of 22:6-monoacylglycerol product ion derivatives. NLS of 345 amu represents the loss of NH₃ and 22:6 fatty acid which is absent for vinyl ether diglyceride species with 22:6 fatty acid at sn-2. Collectively, these tandem mass spectrometric analyses prove the presence of 16:0–22:6 vinyl ether diglyceride species in the studied mouse heart.

Scheme 1

Structure of representative molecular species of three subclasses of diglycerides