Simple LC-MS Method for Differentiation of Isobaric Phosphatidylserines and Phosphatidylcholines with Deuterated Mobile Phase Additives

Abstract

Lipids from different classes sometimes can exhibit the same exact mass upon electrospray ionization; this presents an analytical challenge in lipidomics. In the negative ionization mode, for example, this can occur with phosphatidylcholines (PCs) and phosphatidylserines (PSs), making them indistinguishable in the absence of fragmentation data. PSs are found at low concentrations in biological samples, making MS/MS spectra difficult to obtain. Moreover, while PCs and PSs are distinguishable in the positive mode, PSs do not ionize as well as PCs, and their ionization is suppressed by the PCs. Here, we show that, in the negative ionization mode, substituting protiated LC-MS additives with their deuterated forms provides a way to distinguish PCs and PSs without chemical derivatization. The method described leverages the differential ionization mechanism of PCs and PSs. PCs are ionized via adduction with salts, whereas PSs ionize via hydrogen abstraction. Substituting the salts used for LC-MS with their deuterated form shifts the mass of PCs by the number of deuterium atoms in the salt, while the mass of PSs remains the same. This comparative shift enables their direct differentiation. We demonstrate that the use of deuterated formate shifts the mass of PCs and provides a direct method to distinguish PCs and PSs, even at biologically-relevant low concentrations. The utility of the method was established and validated in the simultaneous analysis of PCs and PSs in lipid extracts from isolated liver mitochondria in two different rat strains. Thirteen low concentration PSs were identified that would otherwise not have been distinguishable from low concentration PCs.

Trimethylation Enhancement Using 13C-Diazomethane: Gas-Phase Charge Inversion of Modified Phospholipid Cations for Enhanced Structural Characterization

“…Structurally informative peaks pertaining to the fatty acyl chains attached to the glycerol backbone are known to be obtained from CID of the negatively charged glycerophospholipids. 13,14,15,19,20,22,23,24 Conventionally, solution additives, such as ammonium acetate can be used to generate adduct anions in the negative mode. A single acetate adduction at the quaternary ammonium site neutralizes the 13 C-TrEnDi-modified species and there are no other sites for the adduction of a second acetate.…”

“…5,12,13,14 However, these gas-phase adducts often have low stability and can inadvertently form isobaric species when applied to complex mixtures. 15,16,17,18 An alternative approach has been to derivatize PLs prior to ionization both to improve the ionization responses of PE and PS lipids in the positive mode 2,19 and to facilitate the distinction of isobaric species 10,12,16 . One such derivatization technique is trimethylation enhancement using 13 C-diazomethane ( 13 C-TrEnDi) modification.…”

“…Solution additives have been used in negative ESI of these PL classes to provide a one-shot method as well as structurally informative fragmentation via tandem mass spectrometry. Acetate or formate salts, for example, have been used to enhance the ionization efficiencies of SM and PC lipids in the negative mode, since collision-induced dissociation (CID) of anionic PL species provides structurally informative fragments and simple tandem mass spectra. ,− However, these gas-phase adducts often have low stability and can inadvertently form isobaric species when the method is applied to complex mixtures. − An alternative approach has been to derivatize PLs prior to ionization, both to improve the ionization responses of PE and PS lipids in the positive mode , and to facilitate the distinction of isobaric species. ,, One such derivatization technique is trimethylation enhancement using 13 C-diazomethane ( 13 C-TrEnDi) modification. TrEnDi covalently modifies carboxylate, phosphate, and primary amine functional groups via 13 C-methylation.…”

Imaging regiospecific lipid turnover in mouse brain with desorption electrospray ionization mass spectrometry

“…Improved identification of lipids is an important direction for future development in biochemistry because of their roles in metabolism and signaling. Identification of lipids is difficult; grouping mass spectral features into (sometimes broad) lipid classes is the Supplemental Material can be found at: current state of the art (15,16). For this proof-of-principle study, we monitored the turnover of four previously identified lipids, but the techniques can be applied to any identifiable molecule.…”

“…Changes in lipid types or concentrations allow boundaries between tissues, as well as regions within the tissue, to be identified (7)(8)(9)(10)12). The spectra from modern highresolution mass spectrometers are exceptionally information rich; but even so, positive identification of all ions in a spectrum is rarely possible and it is often difficult to unambiguously assign peaks to individual lipids (15,16). As a result, many studies have been published using changes in unidentified features to differentiate between areas of an image (8,9).…”