Near-Complete Structural Characterization of Phosphatidylcholines Using Electron Impact Excitation of Ions from Organics

Abstract

Although lipids are critical components of many cellular assemblies and biological pathways, accurate descriptions of their molecular structures remain difficult to obtain. Many benchtop characterization methods require arduous and time-consuming procedures, and multiple assays are required whenever a new structural feature is probed. Here, we describe a new mass-spectrometry-based workflow for enhanced structural lipidomics that, in a single experiment, can yield almost complete structural information for a given glycerophospholipid (GPL) species. This includes the lipid's sum (Brutto) composition from the accurate mass measured for the intact lipid ion and the characteristic headgroup fragment, the regioisomer composition from fragment ions unique to the sn-1 and sn-2 positions, and the positions of carbon-carbon double bonds in the lipid acyl chains. Here, lipid ions are fragmented using electron impact excitation of ions from organics (EIEIO)--a technique where the singly charged lipid ions are irradiated by an electron beam, producing diagnostic product ions. We have evaluated this methodology on various lipid standards, as well as on a biological extract, to demonstrate this new method's utility.

Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO)

“…However, the identification of the specific locations of these carbon-carbon double bonds is much more difficult, requiring the use of ion/molecule reactions (11)(12)(13)(14). With EIEIO, we are able to perform this identification in TAG molecules much the same as we previously reported for PCs (18) (Figs. 2, 3, and 4; supplemental Figs.…”

“…Also appearing in the EIEIO mass spectrum, in the m/z region between the intact TAG precursor ion and the single acyl chain loss product ion, one can find a complete series of acyl chain fragments. Similar to our previous studies (18,19), each carbon-carbon single bond cleavage in the acyl chains was represented by two ions: an even-electron (nonradical) synthesized TAG standards using TAG A/B/C nomenclature given the known composition of such species.…”

“…The detail of ionization method on lipid analysis using a Turbo V TM ion source (Sciex) was described previously (18). Infused flow rate of working solution in this work was 0.3 ml/h, and ESI voltage was +5,500 V. The detail of DMS (SelexION ® Technology, Sciex) for lipid application was described before (19,22).…”

“…In order to acquire more in-depth structural information for lipids, we recently developed a near-complete structural analysis using electron-induced dissociation (16) or electron impact excitation of ions from organics (EIEIO) (17) for phosphatidylcholines (PCs) (18) and SMs (19). Singly protonated precursor lipid ions generated by an ESI were introduced into a branched ion trap (20) wherein they were reacted with a 10 eV electron beam.…”

“…In previous studies (18,19), we have demonstrated the utility of EIEIO for providing detailed structural information for different types of polar lipids (i.e., bearing polar head groups). However, TAGs are somewhat different that these lipids, as they are nonpolar, bearing no easily ionizable moiety themselves.…”