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

Abstract

Methylation of phospholipids (PL) leads to increased uniformity in positive electrospray ionization (ESI) efficiencies across the various PL sub-classes. This effect is realized in the approach referred to as “trimethylation enhancement using 13C-diazomethane” (13C-TrEnDi), which results in the methyl esterification of all acidic sites and the conversion of amines to quaternary ammonium sites. Collision induced dissociation (CID) of these cationic, modified lipids enables class identification by forming distinctive head-group fragments based on the number of 13C atoms incorporated during derivatization. However, there are no distinctive fragment ions in positive mode that provide fatty acyl information for any of the modified lipids. Gas-phase ion/ion reactions of 13C-TrEnDi-modified PE, PS, PC, and SM cations with dicarboxylate anions are shown to charge-invert the positively charged phospholipids to the negative mode. An electrostatically-bound complex anion is shown to fragment predominantly via a novel head-group dication transfer to the reagent anion. Fragmentation of the resulting anionic product yields fatty acyl information, in the case of the glycerophospholipids (PE, PS, and PC), via ester bond cleavage. Analogous information is obtained from modified SM lipid anions via amide bond cleavage. Fragmentation of the anions generated from charge inversion of the 13C-TrEnDi modified phospholipids was also found to yield lipid class information without having to perform CID in positive mode. The combination of 13C-TrEnDi-modification of lipid mixtures with charge inversion to the negative ion mode retains the advantages of uniform ionization efficiency in the positive ion mode with the additional structural information available in the negative ion mode without requiring the lipids to be ionized directly in both ionization modes.

Chemical Tagging in Mass Spectrometry for Systems Biology

“…The fragmentation patterns of the products also held strong structural information. Substantial benefit can be obtained using TrEnDi, though safety considerations are a factor in its use. ,− …”

Enhancing detection and characterization of lipids using charge manipulation in electrospray ionization-tandem mass spectrometry

“…Charge Inversion-While the above approaches effectively transform cations into structurally informative anions by doping chemical additives into lipid solutions, similar strategies have been developed to charge-switch lipid cations in the gas phase. Recently, the McLuckey group 137,[144][145][146][147][148] and others 149 have explored the utilization of gas-phase ion/ion reactions for the charge inversion of lipid cations. For example, Stutzman et al described the charge inversion of PC monocations via ion/ion reaction with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA) anions in efforts to increase structural characterization of PCs.…”

“…In an additional example, gas-phase ion/ion reactions of 13 C-diazomethanemodified PE, PS, PC, and SM cations with PDPA reagent dianions were shown to chargeinvert the positively charged GPL ions to the negative mode, where fatty acyl composition could be revealed. 147 Additional details regarding trimethylation enhancement using 13 Cdiazomethane ( 13 C-TrEnDi) are provided below. The combination of 13 C-TrEnDi modification, PB derivatization, and PDPA reactions in a single workflow permitted enhanced structural identification at the carbon-carbon double bond positional level, particularly for PE lipids in complex biological samples.…”

“…156 However, the CID spectra of TrEnDi-modified lipid cations in positive ion mode do not contain product ions that permit fatty acyl identification, though this limitation can be overcome when combined with additional gas-phase derivatizations as described above. 147,148 We note an alternate methylation approach has also been developed to charge-switch anionic GPL species. Here, the phosphate moieties in PI molecular species from biological extracts are rapidly and completely methylated utilizing a one-step trimethylsilyl diazomethane (TMSdiazomethane) derivatization.…”

Recent Developments in Gas-Phase Ion/Ion Reactions for Analytical Mass Spectrometry

“…Combining charge inversion with other techniques, like trimethylation enhancement using 13 Cdiazomethane ( 13 C-TrEnDi) or the Paternò-Büchi reaction, can result in enhanced structural characterization of PC and PE phospholipids. 134,135 In the above cases, charge inversion was used to convert phospholipid cations to structurally informative anions. Recently, gas-phase charge inversion ion/ion reactions have also been applied to convert fatty acid anions to metalated cations.…”