Cryogenic infrared spectroscopy provides mechanistic insight into the fragmentation of phospholipid silver adducts

Tandem mass spectrometry is arguably the most important analytical tool for structure elucidation of lipids and other metabolites. By fragmenting intact lipid ions, valuable structural information such as the lipid class and fatty acyl composition are readily obtainable. The information content of a fragment spectrum can often be increased by the addition of metal cations. In particular, the use of silver ions is deeply rooted in the history of lipidomics due to their propensity to coordinate both electron-rich heteroatoms and C = C bonds in aliphatic chains. Not surprisingly, coordination of silver ions was found to enable the distinction of sn-isomers in glycerolipids by inducing reproducible intensity differences in the fragment spectra, which could, however, not be rationalized. Here, we investigate the fragmentation behaviors of silver-adducted sn- and double bond glycerophospholipid isomers by probing fragment structures using cryogenic gas-phase infrared (IR) spectroscopy. Our results confirm that neutral headgroup loss from silver-adducted glycerophospholipids leads to dioxolane-type fragments generated by intramolecular cyclization. By combining high-resolution IR spectroscopy and computational modelling of silver-adducted fragments, we offer qualitative explanations for different fragmentation behaviors of glycerophospholipid isomers. Overall, the results demonstrate that gas-phase IR spectroscopy of fragment ions can significantly contribute to our understanding of lipid dissociation mechanisms and the influence of coordinating cations. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-022-03927-6.


In-source fragmentation
In-source fragmentation is equivalent to collision-induced dissociation (CID) based on the acceleration of molecular ions and subsequent collisions with a buffer gas. In the present setup, the ions are accelerated after nano-electrospray ionization and collide with residual gas molecules present in the differentially pumped source region. In order to achieve sufficiently high acceleration, the voltages on the source block and the two ring electrode ion guides (IG) are tuned manually for each ion. Typically, the source block and offset of the first ion guide are set to a maximum value of 150 V. The ions are thus accelerated starting from a high potential towards the second ion guide and then decelerated by an elevated potential on the endcap of the second ion guide. Both the high offset voltage on the source block and first ion guide, and the higher potential on the endcap of the second ion guide compared to the IG 2 offset, are required for fragmentation.
Typical voltages applied to each part in the source region are shown below. Figure S1. Voltage scheme of the source region to induce in-source fragmentation. Precursor ions are generated by nano-electrospray ionization and accelerated from the source block towards the second ion guide. Typical values employed for the fragmentation of silver-adducted phospholipids are shown in the scheme. The drawing is not to scale. Figure S2. Mass spectra showing in-source fragmentation of silver-adducted phospholipids. a) Chemical structure formulae of the precursors PC(16:0/18:1(9Z)) and PE(16:0/18:1(9Z)). b) Silver adducts of PC(16:0/18:1(9Z)) readily eliminate phosphocholine (183 u). c) By applying steeper voltage differences than in (b), PC(16:0/18:1(9Z)) yields an MS 3 fragment at m/z 575 resulting from the loss of phosphocholine and silver hydride. d) Silver adducts of PE(16:0/18:1(9Z)) undergo neutral loss of phosphoethanolamine (141 u).

Silver coordination sites in model lipids
Coordination sites of Ag + on glycerophospholipid fragments were identified using the coordination site screening implemented in CREST [1] (keywords -protonate -swel Ag+) employing the semi-empirical method GFN2-xTB [2] and default settings. The search was performed on a model lipid truncated to three carbon atoms per acyl residue for both dioxolane and dioxane structures. Several coordination geometries were initially found and subjected to a conformational search in CREST. Selected conformers were then optimized in Gaussian 16 [3] at the PBE0+D3/6-311+G(d,p) [4][5] level of theory including an SDD effective core potential for silver. Harmonic vibrational spectra were calculated at the same level of theory and scaled by a factor of 0.965. Free energies including zero-point correction were calculated at 90 K.
The coordination site screening yielded one energetically most favored geometry for both dioxolane and dioxane structures, in which the silver ion coordinates to the carbonyl oxygen and to the sp 2 carbon of the C=C double bond next to the ring. Coordination of the ring oxygen is energetically less favored and decreases the match with the experimental spectra significantly. Overall, the computed spectra of the model lipid yield an unsatisfactory match with the experimental spectrum of [PE(16:0/18:1(9Z)) + Ag − 141] + , which suggests a major influence of the acyl chain length on the band positions in the IR spectrum. Figure S3. Computed IR spectra of a) dioxolane and b) dioxane model structures truncated to three carbons per fatty acid (3:0/3:0) with different silver coordination sites. The experimental spectrum of [PE(16:0/18:1(9Z)) + Ag − 141] + (gray) is shown above the computed spectra for comparison. In the most stable conformers, the silver ion coordinates the carbonyl oxygen and the C=C double bond adjacent to the ring. Spectra were computed at the PBE0+D3/6-311+G(d,p), SDD (Ag) level of theory, and relative free energies at 90 K refer to the most stable conformer of each structure motif. XYZ coordinates of all computed structures are listed on page 15ff. Figure S4. Computed IR spectra and relative free energies at 90 K of silver-adducted dioxolane and dioxane fragments equipped with hexanoic acid chains (6:0/6:0). The experimental spectrum of [PE(6:0/6:0) + Ag − 141] + (gray) is well modeled by the computed spectra of dioxolane structures. Dioxane structures are energetically disfavored, and their IR signatures do not match the experimental spectrum. The deviating band positions in the computed dioxane spectrum at the bottom is caused by energetically unfavorable interactions between the silver ion and ring oxygen. Spectra were computed at the PBE0+D3/6-311+G(d,p), SDD (Ag) level of theory. XYZ coordinates of all computed structures are listed on page 16ff.     Figure S9. Computed IR spectra and relative free energies at 90 K of allylic dioxolane fragments generated from silver-adducted PC(16:0/18:1(9Z)). The core structures of computed conformers are depicted with truncated lipid chains for visibility. The allylic cation features two diagnostic bands between 1550-1650 cm −1 , which were attributed to cis and trans isomers. The carbonyl stretching frequency is shifted by interactions between the carbonyl oxygen and hydrogen atoms of the lipid chain (marked by asterisks). Spectra were computed at the PBE0+D3/6-311+G(d,p) level of theory and XYZ coordinates of all structures are listed on page 31ff.    S13. Overview scheme combining the computed energetics for the abstraction of silver hydride from silver-adducted dioxolane model fragments ( Figure S11) and cis/trans isomerization of the resulting allylic dioxolane fragment ( Figure S12). Comparison of the activation barriers demonstrates that cis/trans isomerization of the allylic dioxolane fragments after their formation is highly improbable due to the considerable activation energy for rotation around the C=C bond. Figure S14. Tandem MS spectra of silver-adducted dioxolane sn-isomers (m/z 683) and dioxolane double bond isomers (m/z 709). Silver-adducted dioxolanes eliminate silver hydride yielding allylic dioxolane fragments at m/z 575 and 601, respectively. The fragment spectra of sn-and double bond isomers exhibit different ratios of oleic acid in the form of ketene (m/z 371) and carboxylic acid (m/z 389). Ketene formation is suppressed in the 6Z isomer of PC(18:1/18:1).

XYZ coordinates of computed conformers
In the following, XYZ coordinates of all conformers corresponding to the computed IR spectra shown in this work are listed. The structures were optimized at the PBE0+D3/6-311+G(d,p) level of theory with an SDD effective core potential for silver. Each conformer is uniquely identifiable by its name and relative free energy (90 K). The name "conf_x" refers to the position x of the conformer in the CREST output file from the conformer sampling. The charge of all conformers is +1 and the multiplicity is 1.    Figure S5 conf_0 0.0 kJ mol −