Lipidome Analysis Essay

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Due to the high diversity of lipid species, their analysis has been challenging. Initial studies on lipid metabolism were often limited to the analysis of individual lipid classes, using techniques with low sensitivity and resolution, such as thin layer chromatography (TLC). Although still applicable due to low costs and rapid processing, progress in lipid research was driven by the development of MS-based techniques. Within the last decade, many different approaches have been developed and successfully applied to basic and clinical research. Untargeted approaches, covering a large fraction of the lipidome, and targeted approaches, focusing on specific lipid classes and species were successfully applied to discover biomarkers for a variety of diseases, such as cancer, metabolic diseases (discussed below) and cardiovascular disease and to decipher molecular mechanisms underlying lipid metabolism in health and disease. In general, three major lipidomics approaches emerged: (i) direct-infusion lipidomics; (ii) MS coupled with chromatographic separation; and (iii) MS imaging. Here we provide an overview of general procedures and concepts in lipidomics, mainly focusing on direct-infusion- and chromatography-coupled MS techniques relevant for research on metabolic disorders. For further information on MS imaging, we refer the reader to recent reviews [17,18,19].

2.1. Preanalytics

The rapid development of MS-based lipidomics approaches allows the analysis of a plethora of sample materials. The lipidome of cultured cells, covering species from yeast to humans, have been analyzed [16,20]. With the entrance of lipidomics into the field of clinical research, lipidomics techniques were applied to a variety of different tissues and body fluids. Furthermore, the analysis of lipids in subcellular fractions of tissues, such as muscle and liver, plays an important role in research related to T2D (discussed below). Proper preanalytical procedures, covering sample collection, storage, preparation and extraction, therefore have become fundamental, not only for lipidomics analysis. Special care has to be taken when biopsies from tissues, such as muscle, are taken for lipidomics analysis. Sample material has to be carefully inspected for blood vessels and extramyocellular adipocytes. Strict protocols have to be in place to remove such contaminations as they might dramatically influence subsequent quantification of lipids (Figure 2A,B) [21]. Generally, tissue is rinsed with buffers and extramyocellular fat is removed manually. In order to prevent degradation of lipids, sample preprocessing has to occur in a timely fashion and samples should be frozen in liquid nitrogen immediately. Long term storage and repeated freeze/thaw cycles of plasma and serum samples showed little changes in the fatty acid composition of lipid classes, such as cholesterolesters (CE), TAG, and glycerophospholipids (GL) [22,23,24]. However, stability in more complex matrices such as body tissues, might differently affect analyte stability and needs to be considered when conducting studies involving lipidomics approaches. This issue becomes even more important when different omics-approaches are combined in a study. Especially, when dealing with peptides and proteins utilizing proteomic profiling approaches, highly standardized sample processing (e.g., avoiding freeze/thaw cycles or utilizing inhibitors) is crucial for successful analysis. Internal standards (IS), used for quantification of lipid species are added to the sample at the earliest time point possible to account for altered extraction and ionization efficiency. Lipids are then extracted from biological samples to remove interfering compounds, such as saccharides and proteins. Methods developed by Folch et al. and Bligh and Dyer, using chloroform and methanol as extraction solvent, are widely used [25,26]. In addition, two-step extraction and methyl-tert-butyl ether (MTBE) extraction protocols are available [16,27]. Automated lipid extraction systems have been developed and allow the high-throughput analysis of a large number of samples, commonly accumulating in clinical research studies [20]. Refinement of lipid extracts by solid phase extraction (SPE) to separate, e.g., lipid classes prior to analysis, is time-consuming, however, further reduces the risk of ion suppression. Thus, lipidomics analyses comprise a comprehensive set of sequential processes, each tailored for specific applications, that are integrated into an overall methodological workflow (Figure 2C). In order to cover more than one class of molecules in a single workflow, very recently, SIMPLEX (simultaneous metabolite, protein, lipid extraction) was introduced to allow a simultaneous and quantitative analysis of lipids, metabolites and proteins derived from one sample [28]. This strategy, addressing different molecular classes in parallel, paves the way to understand the interaction between lipid metabolism and protein driven signaling processes.

2.2. Mass Spectrometry Based Lipidomics

2.2.1. Direct-Infusion MS Techniques

In direct-infusion mass spectrometry, samples, i.e., lipid extracts from biological matrices, are infused into the MS without pre-separation. Described as shotgun lipidomics, this platform was initially developed by Gross and Han, and allows fast and reproducible analysis of lipids in samples [29]. Using a multiplexed lipid extraction, lipid classes can be separated in-source, to enhance performance [30]. Ion suppression caused by those complex sample matrices, especially affects low abundant lipid species and presents a disadvantage of this approach. The advantage of measuring hundreds of lipids in usually limited sample material, however, explains the widespread use of shotgun lipidomics in recent years [16,31,32,33].

2.2.2. MS Coupled with Chromatographic Separation

The chromatographic separation of complex biological samples prior to the MS analysis is widely used to overcome the risks of ion suppression and improve the resolution of isobaric lipid species. Besides TLC, gas chromatography (GC) presents a powerful tool in lipid analytics, still well accepted in fatty acid profiling [34]. Derivatization of lipids, prior to GC analysis, however, might diminish structural information of more complex lipid species. Furthermore, as most lipid species are non-volatile and tend to degrade at high temperatures used in GC, liquid chromatography (LC) systems, coupled to MS detection emerged as powerful tools in lipidomics applications [35,36]. Besides commonly used high performance liquid chromatography (HPLC) systems with normal and reverse phase columns, UHPLC (ultra high performance chromatography), operating at pressures up to 15,000 psi now provide better mass resolution, enhanced sensitivity and greater signal-to-noise ratio [37,38,39]. The chromatographic separation of lipid species reduces ion suppression by reducing the number of competing analytes, entering the MS system at the same time. As retention times for individual lipids are highly reproducible in a given application, methods can be specifically tailored to optimize the analysis time available for individual lipid species. Internal standards for quantification of lipid species have to be selected carefully in order to allow absolute quantification. Due to changing conditions in commonly applied LC gradients, differences in the ionization efficiency of analyte and IS can occur when they elute at different retention times, ultimately affecting quantification results. Finally, carry-over effects on the column can severely affect results, especially when analyzing large numbers of complex biological samples in a row. Methods for the analysis of selected lipid classes and species therefor need to be thoroughly validated in order to account for the possible sources of errors mentioned above.

2.2.3. Identification of Lipid Species by MS

The unequivocal identification of isobaric lipid species presents a major challenge in lipidomics applications. The use of single mass analyzers, e.g., for the analysis of fatty acids by GC-MS, is therefore not applicable for most, more complex, lipid species. Triple quadrupole (QqQ) mass spectrometers were essential for progress in the field of lipid research and were used for direct-infusion shotgun, as well as for chromatography coupled lipid analysis. The system consists of two mass filters, quadrupoles Q1 and Q2, and a collision cell (Q3). Precursor ions passing the first quadrupole undergo fragmentation in the collision cell (collision induced dissociation, CID) and fragments can be analyzed in the third quadrupole. The linear setup of quadrupoles Q1–3 allows different scan modes for the identification of individual lipid classes and subclasses based on their specific fragmentation behavior (Figure 3).

  • Product Ion Scan: fragments of precursor ions, selected in Q1, are analyzed in Q3 after fragmentation in Q2. It is commonly used to study the fragmentation patterns of lipids.

  • Precursor Ion Scan (PIS): in this scan mode, precursor ions, which produce a specific, selected fragment ion (Q3), are detected. Since certain lipid classes have common structural motifs, detected as fragment ion in Q3, PIS can be used to distinguish and identify lipid species within them.

  • Neutral loss (NL): precursor ions with a specific mass difference between the two mass analyzers Q1 and Q3 are detected. The loss of a fragment, corresponding to a lipid class-specific structural motif, is commonly used to identify lipid species of that particular class.

  • Selected reaction monitoring (SRM): SRM is widely used in targeted lipidomics applications because of its high specificity and sensitivity. Selected precursor ions and specific fragment ions are defined for individual lipid species and allow their identification. Multiple reaction monitoring (MRM) is used to analyze multiple lipids. Optimal transitions between precursor and fragment ion are usually determined experimentally, therefore requiring access to reference substances.

Multiple, sequential cycles of fragmentation and product ion analysis of a single precursor ion in MSn experiments can be performed on ion trap mass spectrometry platforms. They provide more detailed structural information on complex lipid species [40,41].

Hybrid quadrupole time-of-flight (QqTOF) mass spectrometers have been used in shotgun lipidomics approaches to overcome limitations of QqQ instruments, which only allow the acquisition of a single PIS or NL per scan. Multiple precursor ion scanning (MPIS) on QqTOF instruments allows the simultaneous acquisition of up to 50 PIS and was applied to characterize the cellular lipidome [20].

The mass spectrometry platforms introduced here, can be combined with a variety of different ionization techniques. Electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) present soft ionization technologies that are routinely used and reduce the risk of in-source fragmentation. In summary, the rapid technological development of mass spectrometry-based technologies provides a comprehensive toolkit to establish tailored solutions for the analysis of lipids, a class of highly divers and biologically and clinically relevant metabolites.

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