Currently, mass spectrometric analysis of lipids in lipidomics is compromised of two complimentary approaches which employ either direct infusion (shotgun lipidomics) or use liquid chromatographic separations prior to mass spectrometric analysis. Shotgun lipidomics is a powerful approach for the analysis of lipids directly from extracts of biological tissues or fluids. Shotgun lipidomics exploits the chemical and physical properties of lipids to facilitate the high throughput global analysis of a cellular lipidome directly from organic extracts of biological samples (Han and Gross, 2005b).
A major advantage of shotgun lipidomics is that a mass spectrum displaying molecular ions of individual molecular species of a class of interest can be acquired at a constant concentration of the lipid solution during direct infusion. This unique feature of shotgun lipidomics allows researchers to perform precursor-ion scans of the particular fragment ions and/or neutral loss scans of the interested neutrally lost fragments for identification and quantitation of the individual molecular species of a lipid class or a category of lipid classes without the time constraints typically encountered through “on the fly” analysis during chromatographic elution. Each suite of scans determines the molecular identity of the molecular ion by recognizing that majority lipid species represent linear combinations of a handful building blocks (Yang et al., 2009). Typical building blocks include a glycerol, sphingoid bases, polar head groups, and fatty acyl substituents (or other aliphatic chains).
There are at least three different types of shotgun lipidomics approaches commonly employed. These include:
1. Quantitation of lipid molecular species of a class by scanning a characteristic fragment diagnostic of the class. Each of the most lipid classes contains one or more characteristic fragments after collision-induced dissociation, which can be used to determine the presence of individual molecular species of the class through precursor-ion or neutral loss scanning or both after direct infusion. Based on these characteristic precursor-ion or neutral loss scan(s), a method for quantitative analyses of these lipid classes after ESI has been developed (Brugger et al., 1997; Welti and Wang, 2004). In this methodology, at least two molecular species of a lipid class of interest need to be added as internal standards to the lipid extracts during extraction of biological samples. These internal standards are used to correct multiple experimental factors for accurate quantification. The selection of these internal standards should consider at least two criteria: (1) these compounds should be absent or present in very minimal amounts in the original lipid extracts; and (2) these compounds should faithfully represent the physical properties (e.g. acyl chain length and unsaturation) of the examined lipid class. Then, a tandem mass spectrometric analysis in either neutral loss or precursor-ion mode can be performed to detect and quantify the individual molecular species of the class in the infused lipid solution. The selected neutral loss or precursor-ion scan, detecting a neutral fragment or a fragment ion should be characteristic of, if not entirely unique to the lipid class of interest.
2. Identification and quantification of individual lipid molecular species by shotgun lipidomics using tandem mass spectrometry. This method employs the high mass accuracy/high mass resolution mass spectrometers, particularly the quadrupole-time-of-flight type of instrument (Ejsing et al., 2006; Ekroos et al., 2002; Schwudke et al., 2006; Schwudke et al., 2007; Ståhlman et al., 2009). By using this type of instrument, a product ion spectrum of each molecular ion in a unit mass after direct infusion can be rapidly and efficiently acquired (Ståhlman et al., 2009). After performing product ion analyses of molecular ions within a mass range of interest, any interested precursor-ion scans and/or neutral loss scans can be extracted from the detected product ions. The analyses can be conducted in both positive-ion and negative-ion modes in the presence of ammonium acetate in the infused solution. Identification can be performed from bioinformatic reconstruction of the fragments from precursor ion or neutral loss scans. Quantification can be achieved through comparison of the sum of the intensities of extracted fragments of an ion to that of a pre-selected internal standard.
3. Identification and quantification of individual lipid molecular species by multi-dimensional mass spectrometry-based shotgun lipidomics (MDMS-SL). If we hypothetically ramp the neutral loss of all potential fragments or monitor all potential fragment ions unit by unit in a mass range of interest, each of the ramps in mass or mass to charge, respectively, constitutes a two-dimensional map of the molecular species in the determined mass range. The first dimension is the molecular ions (x-axis) in m/z values, while the second dimension is comprised of the mass corresponding to the neutrally lost fragments or the monitored fragment ions in m/z values (y-axis). The cross peaks of a given primary molecular ion in the first dimension with the second dimension represent the fragments of a given molecular ion. Analysis of these cross peaks (i.e. the individual fragments) thereby identifies the structure of the given molecular ion as well as its isobaric substituents (Han and Gross, 2005a). Han and Gross have referred to these kinds of two-dimensional maps as two dimensional mass spectrometry (2D MS) (Han and Gross, 2001; Han et al., 2004; Han and Gross, 2005a), since they are entirely analogues to two-dimensional NMR spectroscopy. The only difference between these mapping approaches is that the former uses units in the mass domain while the latter uses units in the frequency domain. Since naturally occurring lipids are comprised of known building blocks, this process is readily simplified by monitoring only those building blocks that are characteristic of individual molecular species of a lipid class of interest (i.e. building blocks of the class of interest). In this simplified 2D MS, the y-axis is the building blocks of a lipid class or a category of lipid classes. Individual lipid molecular species of a class of interest by 2D MS can be automatically identified (Yang et al., 2009).
In theory, to investigate extensively the effects of ionization conditions on ionization efficiency and/or the effects of collision conditions on fragmentation processes, a variety of ionization voltages, ionization temperatures, collision energies, collision gas pressures, etc should be employed in a particular experiment. These variables can all be ramped within a certain range. Therefore, each of these variables constitutes an additional dimension to the mass ramp described above. All these dimensions form the family of multi-dimensional MS (Han and Gross, 2005a).
Quantification of the identified individual molecular species of a class of interest is performed in a two-step procedure as outlined previously (Cheng et al., 2007). Specifically, the molecular species of the class of interest that are abundant and not overlapping with species in any other classes are quantified by ratiometric comparison with the selected internal standard of the class by using a full MS scan. Then, some or all of these quantified molecular species plus the original internal standard are employed as standards to quantitate other low abundance and/or overlapped molecular species in the class by using one or more class-specific precursor-ion scans and/or neutral loss scans. Using this two-step methodology, the dynamic range of quantitation can be increased by at least two orders of magnitude as previously demonstrated (Han et al., 2008).
References:
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