Mass Shifting and Radical Delivery with Crown Ether Attachment for Separation and Analysis of Phosphatidylethanolamine Lipids

Abstract

Phosphatidylethanolamines (PE) and phosphatidylcholines (PC) are important phospholipids frequently present in many types of cells. In some cases, PE has been equated with PC because they are chemically very similar and are often isomeric species. In this study, we demonstrate that noncovalent complexation between PE and 18-crown-6 ether (18C6) can be used to quantitatively mass shift and separate PE from PC phospholipids. Detection of PE is also more sensitive by approximately an order of magnitude with addition of 18C6. This noncovalent complexation approach is used to separate and quantitatively characterize PE in a soy bean asolectin extract. 18C6 (modified with an iodobenzoyl moiety) can also be used to efficiently generate radical PE lipids following photoactivation in the gas phase. Subsequent collisional activation of these lipid radical ions leads to radical directed dissociation (RDD), which generates unique fragment ions relative to dissociation of comparable even electron ions. Interestingly, RDD produces fragment ions that reveal carbon bonding features within the lipid acyl chain substituents, such as double bond location or the presence of branching. Furthermore, several novel and abundant fragments were observed in unsaturated lipids. Mechanisms that can account for the high abundance of some of these product ions are proposed.

Electron Transfer Reduction of the Diazirine Ring in Gas-Phase Peptide Ions. On the Peculiar Loss of [NH4O] from Photoleucine

“…In particular, CID of (GL*GGR +2H) +• showed dominant m/z 438 (ion A, loss of [NH 4 O]) and m/z 442 (ion B, loss of N 2 H 2 ) fragment ions, whereas amongst the sequence fragment ions only the z 3 (m/z 273) and y 3 (m/z 289) were formed competitively ( Figure 2c). Note that the z 3 ion is accompanied by a metastable x 3 ion at m/z 316 [40][41][42], indicating that this radical-triggered backbone dissociation occurs in two steps [42].…”

Re-print of “Radical Delivery and Fragmentation for Structural Analysis of Glycerophospholipids”

“…Fresh lipid samples were prepared by diluting chloroform stock solutions or lipid extracts in methanol to a final concentration of 1-5 M. When desired, IB-18C6 (with a ratio lipid:crown 1:4) was added to solution immediately prior to positive ion ESI-MS experiments [25].…”

“…Radical-based dissociation techniques have been used to provide unique information in experiments targeting peptides and proteins, [14][15][16][17][18][19][20] and these radical-based methods have recently DOI started to attract attention in the investigation of lipids as well [21][22][23][24][25]. One route for generating radical species is to use electrons as in electron capture dissociation (ECD) or electron-transfer dissociation (ETD).…”

“…Generation of lipid radicals in this fashion was used to successfully discriminate between branched and non-branched phosphatidylcholine (PC) lipid isomers [23]. Alternatively, 4-iodobenzoyl 18-crown-6 (IB-18C6) interacts very specifically with primary amine containing lipids such as those with phosphoethanolamine (PE) and phosphoserine (PS) headgroups [25]. In this case, noncovalent attachment of 18C6 is so efficient that it can quantitatively mass-separate PE lipids from their PC isomers in biological samples.…”

“…In this case, noncovalent attachment of 18C6 is so efficient that it can quantitatively mass-separate PE lipids from their PC isomers in biological samples. A novel product ion from RDD in experiments with IB-18C6 was used to determine the location of the double bond for lipids with an unsaturated fatty acid chain [25].…”