Experimental evidence that electrospray-produced sodiated lysophosphatidyl ester structures exist essentially as protonated salts

Introduction

The site(s) of ionization of small multi-functional molecules such as glycerophospholipids (GPLs) relies essentially on the analyte interaction with ionized agents as organic mobile phase additives (e.g. ammonium) or more generally with naturally occurring alkali cations (e.g. sodium) under positive electrospray conditions. During aggregate desolvation, the formed GPL adduct ions may either survive as a hydrogen-bonded heterodimer with organic cation or dissociate through proton exchange into protonated molecules. ¹ On the other hand, alkali adduct ions may survive with structures being either a charge-solvated^2–4 or protonated salt.^2,5,6 These processes highly depend on the structures of GPL molecules within charged droplets/aggregates. Indeed, such multi-functional species might exist either or both under (i) canonical or (ii) zwitterion form(s). ⁶ These forms can be used to describe the structure(s) of lysoglycerophosphatidylethanolamine (LGPE, Scheme 1(a)) and lysoglycerophosphatidylcholine ester molecules (LGPC, Scheme 1(a)). Due to the presence of a trimethyammonium group within their structure, LGPC species only exist as betaine structures (i.e. a zwitterion-like form).^2,6 Such neutral species would be ionized during the last step of the charged aggregate desolvation. A preliminary attempt for rationalizing differently ionized (protonated and sodiated) GPL molecular species was published recently. ⁶ A still pending question regarding cationized forms is how the cation is bound to lipids, either as a cation-solvated or as a protonated salt.

The protonation of the anionic phosphatidyl site from the zwitterion form of the LGPE results mainly in a protonated canonical form with an ammonium site from which the proton can be readily mobilized. In the opposite, the protonation of LGPC species would yield ionic molecular species with a permanent positive charge that might form a charged salt bridge upon structure refolding (Scheme 1(b)).

In a similar way, sodiated LGPE species in gas phase might exist as a charge-solvated structure when considering the canonical form and/or as a protonated salt structure from the zwitterion form (Scheme 1(c)). ⁶ In the literature, these cationized molecules are mainly considered as charge-solvated forms^7–10 rather than protonated salts.^3,5 Reasons for such a choice are often not discussed and as a consequence, proposed mechanisms to explain subsequent ion dissociation under low collision energy are generally considered as occurring by charge-remote fragmentation processes (i.e. the charge is spectator of the dissociation). However, such dissociation type cannot be envisaged under low collision energy conditions except when considering metal complexes with metal reduction occurring under activation. ¹¹ In the case of LGPC bearing a permanent positive charge, the deprotonated phosphatidyl moiety would interact preferentially with Na⁺, although the charge-solvated form a priori could not be ruled out (Scheme 1).

In order to interpret the product ion spectra of sodiated LGPLs, under low collision energy conditions in the positive ionization mode, it is essential to determine if either one or both canonical or zwitterionic forms are generated in the electrospray-ionization (ESI) source. For this purpose, we investigated the decompositions of the LGPE (e.g. LGPE 13:0) and LGPC (e.g. LGPC 11:0) sodiated forms using a high-resolution hybrid tandem quadrupole time-of-flight (Q/TOF) instrument at a selected m/z range that allows to detect low m/z ratios in order to highlight the presence of the diagnostic sodium cation and other low m/z product ions.

Material and methods

Results and discussion

The distinction between charge-solvated and protonated salt forms of [M+Na]⁺ can be reached by the detection of Na⁺ as diagnostic product ion of the charge-solvated structures. Protonated species should present drastically different product ion spectra than the charge-solvated species due to the preferential cleavage of covalent bonds. In order to improve the sodium cation transmission and detection, the experimental conditions were thoroughly optimized using sodiated PEG 600 known to occur essentially as cation-solvated form.^13,14 After optimization, the low-energy CID spectrum of sodiated PEG displayed exclusively Na⁺ as product ion (Figure S1, Supplementary material). LGPE and LGPC species were then investigated under these optimized conditions.

The mass spectra of LGPE 13:0 and LGPC 11:0 both display a prominent [M+Na]⁺ ion at m/z 434.228 (Figure S2a) and m/z 448.244 (Figure S2b), respectively. Corresponding protonated molecules from LGPE 13:0 and LGPC 11:0 detected at m/z 412.246 and 426.262 appear comparatively at a very low abundance (less than 5%) comparable to those of cationized molecule with potassium at m/z 450.201 and m/z 464.215, respectively.

Under low collision energy conditions (E_lab = 35 eV, Figure 1(a)), activated dissociations of sodiated LGPC 11:0 (m/z 448.244) give rise to the formation of an intense production at m/z 389.171 (base peak). To a lesser extent, the m/z 243.196 ion (20% of base peak) is also detected. Additional fragments at m/z 104.107 and m/z 86.096 also appear with very weak abundances (i.e. less than 4% of m/z 389.171). These fragment ions probably result from covalent bond dissociations promoted first by the direct loss of trimethyl amine (C₃H₉N) to yield the ion at m/z 389.171, which then exhibits competitive losses of C₂H₄O ethylene oxide and sodiated ethylene phosphate C₂H₄PO₄Na salt yielding fragment ions at m/z 345.143 and m/z 243.196, respectively. Complementary MSⁿ experiments performed under resonant conditions on a tribrid Orbitrap Fusion further confirmed these dissociation pathways (Figure S3, see Supplementary material). Of note, the ions at m/z 104.107 and m/z 86.096 were not detected in any of these experiments, thus meaning that these species might be generated through distinct fragmentation pathways.

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Figure 1.

CID spectra (E_lab = 35 eV) of the selected sodiated [M+Na]⁺ ion from LGPC11:0 (m/z 448.244) (a) and corresponding ERMS breakdown curves where only the most abundant product ions are considered (b).

Interestingly, although optimization of the low m/z ion transmission was done (as for PEG 600, see Supplementary material), the Na⁺ product ion appears at a much higher energy compared to the other fragment ions at m/z 389.171 and m/z 243.196 resulting from covalent bond cleavages. Indeed, apparition threshold of Na⁺ occurs at around an E_lab value of 30 eV as deduced from the ERMS breakdown curves of the m/z 448.2445 dissociations (Figure 1(b)) and beyond this value, its abundance increases until being the most intense product ion for E_lab > 55 eV. This behavior contrasts with what can be observed for the CID spectrum of the sodiated PEG600 species that are charge-solvated structure (Figure S1b). Such a particular behavior implies that the sodiated LGPC 11:0 cannot exist predominantly as a charge-solvated structure. Indeed, the introduction of Na⁺ as charge-solvated form (by the neutral ester and hydroxyl groups) in addition to the presence of the two opposite charges of the betaine moiety, must be very energetically unfavorable (Scheme 1(d)).

Under the same low collision energy conditions (E_lab = 35 eV, Figure 2(a)), sodiated LPGE (m/z 434.228) dissociates mainly to yield the m/z 391.186 and m/z 271.227 fragment ions by the aziridine (C₂H₅N) loss with sodium retention within the fragment ion, and by sodiated phosphoethanolamine salt release, respectively. Those ions are generated by covalent bond cleavages at the polar head-end group without the detection of the naked Na⁺ cation. This can be deduced from the ERMS breakdown curves of the selected m/z 434.228 precursor ion which is almost completely absent at E_lab > 25 eV whereas Na⁺ only begins to be visible beyond E_lab = 30 eV (Figure 2(b)), similarly to the LGPC lipid species (vide supra). Na⁺ becomes the base peak ion for E_lab > 50 eV. The covalent cleavage at low excitation energy seems specific to the protonated salt structure for sodiated LGPE 13:0. In addition, the late appearance of the Na⁺cation is not an indication of a charge-solvated structure generated from LGPE in a canonical form. Consequently, zwitterionic structure (Scheme 1(a)) should better characterize the LGPE molecule with sodium attachment occurring at the phosphatidyl anion site. In addition, the m/z 434.228 precursor ion might be viewed as a protonated sodiated LGPE salt rather than a charge-solvated form. The latter structure could be generated from the canonical LGPE form although production of such a charge solvated structure (from its canonical form) should be more favored than from LGPC form which is a betaine.

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Figure 2.

CID spectra (E_lab = 35 eV) of the selected sodiated [M+Na]⁺ molecules from LGPE13:0 (m/z 434.228) (a) and corresponding ERMS breakdown curves where only abundant product ions are considered (b).

Both sodiated LGPC and LGPE molecules displayed a similar behavior although the former species carry a permanent positive charge and thus no available mobile proton. Indeed, aziridine and sodiated phosphoethanolamine salt releases can competitively take place from LGPE species through proton migration from the protonated amine to an oxygen atom of sodiated phosphatidyl group, thus promoting covalent bond cleavages. In addition, Na⁺ cation retention occurs on weakly abundant product ions of small size such as m/z 120.966 and m/z 164.010 ions corresponding to (HO)₂P(ONa)OH⁺ and H₂N(CH₂)₂OP(OH)(ONa)OH⁺, respectively. Their relative abundances increase at higher E_lab values as well as the naked Na⁺ cation which suggests that they are very likely produced through consecutive processes.

Conclusion

In order to perform this study, the Q/TOF instrument was optimized to allow the favored transmission and detection of low m/z product ions such as Na⁺ to demonstrate the formation of sodiated lysoglycerophospholipids under low collision energy. The Na⁺ detection should indicate the survival of charge-solvated forms from aggregate desolvation occurring in the ESI source. Instead, low-energy fragmentation experiments of LGPC and LGPE lipid species showed that only competitive and consecutive covalent bond cleavages take place through proton migration (and not charge-remote dissociations). Such particular behavior provides evidence of the preferential production of the protonated salt structure. This also implies that, under the ESI-positive conditions used, LGPE and LGPC molecules exhibit zwitterion and betaine structures, respectively.

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Scheme 1.

Proposed structures of (a) canonical and zwitterionic forms of LGPE , and betaine form of LGPC, (b) protonated LGPE and LGPC, (c) sodiated LGPE represented as charge-solvated from the canonical form, and as a protonated salt from the zwitterionic form, and (d) sodiated LGPC represented as charge-solvated with salt–bridge interactions and a sodiated phosphate salt from the betaine structure.

Supplemental Material