Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies

Abstract

This article is available online at http://www.jlr.org technique generates charged molecules (i.e., for m/z < 1,000 Da) without signifi cant analyte decomposition ( 4 ). The technique also enables the coupling of HPLC to MS, effectively converting liquid-phase molecules into gasphase ions. Another leap in technology was the introduction of the triple quadrupole mass spectrometer ( 5 ), which when combined with ESI, enables facile, sensitive, and specifi c analyses of complex lipid mixtures ( 6, 7 ). Using ESI-MS, semitargeted scan modes, which exploit unique ion fragmentation patterns during MS/MS experiments, were developed that are presently used to isolate categories and classes of lipids without the need for complex LC separation, a technique termed "shotgun" lipidomics ( 8 ). There are several technical limitations of this approach including restricted detection of minor lipid components due to instrumental constraints of the dynamic range and discrimination against low-abundant and/or poorly ionizable lipids. Furthermore, ion suppression and diffi culty in differentiating lipids with overlapping fragmentation patterns underline the importance of chromatographic separation prior to MS analysis. A practical alternative to a global lipidomics methodology is a targeted lipidomics approach that utilizes extraction and analysis protocols that are optimized for each lipid category and class ( 9 ), providing an effective tool to identify Abstract Phospholipids serve as central structural components in cellular membranes and as potent mediators in numerous signaling pathways. There are six main classes of naturally occurring phospholipids distinguished by their distinct polar head groups that contain many unique molecular species with distinct fatty acid composition. Phospholipid molecular species are often expressed as isobaric species that are denoted by the phospholipid class and the total number of carbon atoms and double bonds contained in the esterifi ed fatty acyl groups (e.g., phosphatidylcholine 34:2). Techniques to separate these molecules exist, and each has positive and negative attributes. Hydrophilic interaction liquid chromatography uses polar bonded silica to separate lipids by polar head group but not by specifi c molecular species. Reversed phase (RP) chromatography can separate by fatty acyl chain composition but not by polar head group. Herein we describe a new strategy called differential ion mobility spectrometry (DMS), which separates phospholipid classes by their polar head group. Combining DMS with current LC methods enhances phospholipid separation by increasing resolution, specifi city, and signal-to-noise ratio. Additional application of specialized information-dependent acquisition methodologies along with RP chromatography allows full isobaric resolution, identifi cation, and compositional characterization of specifi c phospholipids at the molecular level. The modern fi eld of lipidomics began with the advent of ESI-MS [for review, see ( 1-3 )]. This "soft" ionization

In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry

“…1) (10). The fundamental behavior of DMS and its application to lipid analysis are described elsewhere (11)(12)(13). Briefly, the DMS cell is mounted in the atmospheric pressure region between the ESI source (held at 5,000 V) and the spectrometer's sampling orifice.…”

“…quadrupole TOF mass spectrometer with a differential mobility spectrometry (DMS) cell (SelexION TM Technology) installed between the ion source and the mass spectrometer (11)(12)(13). The DMS was used to isolate SM from other interfering lipid classes, namely PCs and triacylglycerols (TAGs), prior to analysis.…”

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

“…1-4), the TAG molecule's structures were successfully analyzed and provided information 20% of the monoisotopic base peak of the interfering TAG, if it is present in a very large excess compared with the targeted TAG, convolution of two TAG EIEIO spectra will occur. Hence, we used DMS (22,26,27) to separate analyte TAGs from species such as interfering isotopomers, oxidized TAGs, and other unknown isobaric interferences. Such efforts reduce complexity of the EIEIO spectra and increase the confidence of the deconvolution process for separating contributions from multiple species with similar molecular structures.…”

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data

“…[84][85][86] This method separates lipid classes before they are subjected to quadrupole selection of molecular ions and then collision-induced formation of product ions, thereby substantially deconvoluting complex overlapping ions. It has been applied to the separation of leukotriene and protectins.…”