Article pubs.acs.org/ac
Near-Complete Structural Characterization of Phosphatidylcholines Using Electron Impact Excitation of Ions from Organics J. Larry Campbell and Takashi Baba* SCIEX, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada S Supporting Information *
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.
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be identified easily in positive-ion mode, systems must be switched to negative-ion mode to provide acyl chain identification using MS/MS.1 Alternatively, ion/ion chemistry can be used to charge invert phospholipid ions from positive to negative forms to accomplish this characterization.4 Beyond identifying a phospholipid’s class and acyl chain constituents, identifying and quantifying the connectivity (or regioisomerism) of the two acyl chains can be challenging. Although simple negative-ion mode fragmentation of many pure phospholipid standards can yield approximate ratios for the acyl chain substituents at the sn-1 and sn-2 positions, the presence of two regioisomers (at the same m/z) make such analyses inaccurate. Generally, such regioisomer knowledge can only be assessed using an MS mode with multiple stages of fragmentation and isolation (e.g., MSn, where n ≥ 3),5,6 a form of lipid ion separation prior to mass analysis either by chromatography7 or ion mobility,8 or the fragmentation of metal-adducted phospholipids.8−10 The final frontier in lipid characterization relates to the identification and location of carbon−carbon double bonds along the substituent acyl chains.11 The first examples of using MS to elucidate double bond positions in ionized lipids employed high-energy collision-induced dissociation (CID)12 integrated into now-supplanted MS technologies (e.g., fastatom bombardment − FAB, sector MS, etc.). Regardless, many of these MS methodologies are capable of subjecting lipid ions to very high center-of-mass collision energies (≥keV), resulting
n recent years, mass spectrometry (MS)-based methodologies have provided great advances in the molecular characterization of lipids.1 Generally, these lipids are present in complex lipid extracts, which are ionized using electrospray ionization (ESI)2 and then sampled by a mass spectrometer. Their masses (or mass-to-charge (m/z) values) can be measured using good mass accuracy (10 min) using very concentrated solutions (100 μg/mL) provides very weak signals for diagnostic fragment ions. Ion chemistries have also been explored to identify double bonds in lipids. Ozone-induced dissociation (OzID),17−19 a method by which double bonds in lipid ions are oxidized and then identified by the oxidation location, can be combined with traditional MS2 to provide double bond locations and regioisomerism information.6 When coupled with LC, OzID can also provide phospholipid isomer separation and characterization in some cases.7 Other ion/ molecule methodologies exist for double bond identification, such as in-source acetonitrile adduct formation20 and a UVinduced Paternò-Büchi reaction,21 but complex mixtures of precursors and adducts result, complicating true analyte selection. While valuable, each of these MS techniques require multiple stages of MSn or different instruments entirely for each separate level of lipid characterization. As an alternative, we have developed a lipid characterization workflow that provides near complete structural characterization of phospholipid ions in a single experiment. We employ electron impact excitation of ions from organics (EIEIO)22 or electron-induced dissociation (EID)23,24 to generate fragmentation patterns for phospholipids that reveal Brutto composition, headgroup identification, acyl chain substituents, their regioisomeric orientation, and the presence and location of carbon−carbon double bonds in one experiment. Like other examples of EIEIO/EID fragmentation of singly charged ions,25−28 our experiments yield additional structural information not observed in collision-induced dissociation spectra. The mass spectrometer we employeda modified hybrid quadrupole time-of-flight mass spectrometer (qTOF-MS)contains an electron-capture dissociation (ECD) cell29 that switches easily from proteomics to these lipidomics applications by simple alteration of instrument voltages and timing schemes.
Table 1. Ion Source Parameters Employed during the EIEIO Experiments parameters
settings
ion spray voltage source temperature curtain gas nebulizer gas desolvation gas
+5500 V 25 °C 10 psi 10 psi 0 psi
Instrument Control and Data Analysis. The mass spectrometer (including the ECD parameters and data acquisition) was controlled using research-grade software developed in-house. EIEIO mass spectra were visualized using PeakView 1.2 (SCIEX), whereas data analysis, including identification of phospholipid class, regioisomerism, and double bond location, was performed using an interface developed in C# using Visual Studio (Microsoft Redmond, WA). Available Modes for Performing EIEIO of Lipids. Lipid ions were isolated using the first mass resolving quadrupole (Q1) and were subjected to one of two possible EIEIO experiments. In the trapping-mode experiment, lipid ions were injected into the reaction region for between 5−40 ms. Then, these trapped ions were irradiated with an electron beam (KEe = 9−10 eV) for 20 ms, after which time the electron beam was turned off; all ions−intact and fragmented lipid species−were ejected from the reaction region toward the TOF for mass analysis. This trapping mode was used to analyze the synthesized PCs and the most abundant PC species in the egg yolk extract. The second available EIEIO experiment involved simultaneous trapping of both the lipid ions and electron beam.29 Here, lipid ions were injected for 20 ms, during which time the electron beam was engaged. Then, all ions were ejected from the reaction region (1 ms) for mass analysis. The sensitivity of this mode is higher than trapping mode and was used for analyzing the less abundant species in the egg yolk extract. Accumulation times for the EIEIO-TOF experiments ranged from 18 min for the least sensitive species in the egg yolk sample (e.g., m/z 744, see Figure SI-5), while accumulation times of
