Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria. <s> BIB001 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometry method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C 8 column and a binary gradient, more than 160 lipids belonging to eight different classes were detected in a single LC-MS run. The method was fully validated and the analytical characteristics such as linearity (R 2 , 0.994-1.000), limit of detection (0.08-1.28 μ/mL plasma), repeatability (RSD, 2.7-7.9%) and intermediate precision (RSD, 2.7-15.6%) were satisfactory-The method was successfully applied to p53 mutant mice plasma for studying some phenotypic effects of p53 expression. © 2008 American Chemical Society. <s> BIB002 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> In the studies of lipid metabolomics, liquid chromatography electrospray ionization mass spectrometry (LC/MS) is a robust and popular technique. Although effective reverse-phase LC methods enabling the separation of phospholipid molecular species have been developed, there are still problems with the separation of phosphatidic acid (PA) and phosphatidylserine (PS). These acidic phospholipids often elute as extensively broad peaks, causing inferior separation, detection, and quantification-a severe limitation of the method. In this study, we have developed reverse-phase LC conditions that reduce the undesired peak tailings in the elution profiles of both PA and PS, by using a starting mobile phase containing a low concentration of phosphoric acid (5 microM) and a high percentage of water (40%). Our method sensitively analyzes PA, PS, and their lysoforms, as well as the other phospholipids within a biological sample, in a single chromatographic step by an LC/MS method and, thus, is suitable for lipidomics. <s> BIB003 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> Recently, global analysis of triacylglycerols (TAGs) has become increasingly important in studies of abnormality of lipid metabolism in metabolic syndrome. TAGs consist of various molecular species, caused by their three fatty acyl chains with a large variety of carbon chain lengths and degrees of unsaturation. Therefore, most previously reported methods have been insufficient in global detection of TAGs including their structural isomers and TAGs with oxidized or odd number acyl carbon chain. Here we report an effective method for global analysis of TAG molecular species from complex lipid mixtures of mouse liver and white adipose tissue (WAT) using reverse-phased high resolution liquid chromatography (LC) coupled with electrospray ionization (ESI)-quadrapole/time of flight hybrid mass spectrometer (QTOF-MS). For effective profiling of TAG molecular species, sensitive two-dimensional (2D) maps were constructed and individual structures were correctly identified by the elution profile and MS/MS. As a result, TAGs including their structural isomers and TAGs with an odd number acyl carbon chain were separated and detected effectively on the 2D map as compared with conventional high performance LC. It was also found that our 2D profiling method was useful in searching characteristic molecular species globally. In mouse WAT, novel oxidized TAGs, which were mainly formed by hydroperoxidation of one of their linoleic acyl chains, were effectively detected in comparison with TAG molecular species of mouse liver. <s> BIB004 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> A profiling method for glycerophospholipids (GPs) in biological samples was developed using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (LIT-FTICRMS) with electrospray ionization (ESI) in the negative ionization mode. The method allowed qualitative (identification and structure elucidation) and relative quantitative determination of various classes of GPs including phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, and cardiolipins in a single experiment. Chromatographic separation was optimized by the examination of different buffer systems and special emphasis was paid on the detection by ESI-MS. The hybrid LIT-FTICRMS system was operated in the data-dependent mode, switching automatically between FTICRMS survey scans and LIT-MS/MS experiments. Thereby, exact masses for elemental composition determination and fragmentation data for identification and assignment of fatty acid residues are provided at the same time. The low absolute instrumental limits of detection (0.05 pmol for phosphatidylglycerol to 1 pmol for phosphatidic acid) complemented by a linear dynamic range of 1.5 to 2.5 orders of magnitude facilitated the relative quantification of GP species in a lipid extract from Saccharomyces cerevisiae. The developed method is a valuable tool for in-depth GP profiling of biological systems. <s> BIB005 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversed-phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome. Copyright © 2009 John Wiley & Sons, Ltd. <s> BIB006 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimer's disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimer's disease (AD) patients from those of age-matched healthy controls. <s> BIB007 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> In the present work we describe the development of an analytical technique for simultaneous profiling of over 100 biochemically related lipid mediators in biological samples. A multistep procedure was implemented to extract eicosanoids and other bioactive lipids from the biological matrix, chromatographically separate them using fast reversed-phase liquid chromatography, tentatively identify new candidate eicosanoids through a matching process of retention times, isotope distribution patterns, and high-resolution orbitrap MS/MS fragmentation patterns, and subsequently quantify tentative candidates by means of analytical reference standards. Key new aspects of this profiling technique included the classification of bioactive lipids into 12 groups according to their calculated exact masses and the development of optimized liquid chromatographic conditions for these groups to achieve sufficient separation of the numerous isobaric and isomeric species, many of which exhibited virtually identical collision-ind... <s> BIB008 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> In this paper, analytical methodologies for the global profiling of lipids in serum and tissue samples are reported. The sample preparation is based on a modified Folch extraction, and the analysis is carried out with ultrahigh-performance liquid chromatography combined with mass spectrometry (UPLC-MS). For further identification, MS(n) mass spectrometry is carried out utilizing an LTQ-Orbitrap mass spectrometry as the detector. Such a system affords determination of accurate masses and is thus a highly useful tool for lipid identification. The repeatability of the analysis proved to be good, with relative standard errors for spiked samples being between 4.51 and 10.44%. The throughput of the methodology described here is over 100 samples a day. <s> BIB009 </s> Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms <s> High Resolution Mass Spectrometry <s> This work aims to combine chromatographic retention, high mass resolution and accuracy, MS/MS spectra, and a package for automated identification and quantitation of lipid species in one platform for lipidomic analysis. The instrumental setup elaborated comprises reversed-phase HPLC coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT), and Lipid Data Analyzer (LDA) software. Data analysis for lipid species quantification in this platform is based on retention time, mass resolution of 200,000, and mass accuracy below 2 ppm. In addition, automatically generated MS/MS spectra provide structural information at molecular level. This LC/MS technology allows analyzing complex biological samples in a quantitative manner as shown here paradigmatically for murine lipid droplets having a huge surplus of triacylglycerol species. Chromatographic preseparation of the bulk lipid class alleviates the problem of ion suppression of lipid species from other classes. Extension of 1D to 2D chromatography is possible, yet time consuming. The platform affords unambiguous detection of lipid species as low as 0.1‰ within major lipid classes. Taken together, a novel lipidomic LC/MS platform based on chromatographic retention, high mass resolution and accuracy, MS/MS analysis, and quantitation software enables analysis of complex samples as demonstrated for lipid droplets. <s> BIB010
Quadrupole-TOF mass spectrometry offers several advantages. On one hand, this instrumentation provides resolution of up to 40,000 and mass accuracy of better than 5 ppm, which is sufficient for pinning down many of the elemental compositions encountered in lipidomic analysis. On the other hand, TOF analyzers have a very high scan rate and acquire full product ion spectra very fast and efficient. On the downside is the usually limited dynamic range of the detector, which limits quantitation to a rather narrow concentration range. Nevertheless this kind of instrumentation is a valuable tool when coupled to reversed phase HPLC. Successful application of this experimental setup was used for analysis of TG and oxidized TG species. In this case, it was even possible to determine the actual fatty acid composition of TG molecular species by product ion spectra on all major species BIB004 . An excellent example for an integrated lipidomic platform relying on reversed phase ultra performance liquid chromatography (UPLC) quadrupole-TOF is shown by the group of Oresic BIB009 , whereby a combination of retention time, exact precursor mass and product ion spectra are used for identification of lipids from various lipid classes. In contrast to widely used gradient elution, the group of Wenk present a profiling method based on quadrupole-TOF and isocratic reversed phase HPLC BIB001 used for determination of anionic glycerophospholipids, glycolipids, fatty acids, prenols and sphingolipids. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) offers literally unlimited mass resolution and sub ppm mass accuracy. Its combination with a linear ion trap (LTQ-FT) has become a high end standard instrumentation in proteomic research, but a few groups also use it in lipidomic research. The instrument's hybrid character holds the possibility to run the linear ion trap and the FT-ICR-MS as two instruments in parallel, resulting in high resolution precursor spectra and low resolution product ion spectra at an increased duty cycle. Coupled to HPLC, this experimental platform delivers retention time, exact sub ppm precursor masses and product ion spectra as means for identification (Figure 3 ). High mass accuracy paired with retention time is particularly helpful for identification of lipids with too little intensity for reliable fragment spectra. Such an integrated experimental platform is successfully used for quantitative determination of various lipid classes in lipid droplet preparations BIB010 . MS/MS spectra are triggered in a data-dependent manner on the four most intensive ions in the preview scan, resulting in MS/MS coverage of 66%. Owing to the ultra high resolving power and mass accuracy it is possible to confidently detect lipid species in crude lipid extracts at extremely low quantities, even when no MS/MS spectra are available for the precursor. Extension of reversed phase HPLC by a preceding HILIC fractionation of certain lipid classes results in higher sensitivity for lipid classes previously suppressed by PC. Other successful applications of similar instrumental setup are methods for quantitation of glycerophospholipids and TG in plasma samples BIB002 and for identification of glycerophospholipids in yeast BIB005 . The disadvantages of such systems are their still rather slow duty cycle of about 3 s at 200,000 mass resolution, and the low mass cutoff in the linear ion trap, which might cause loss of some low mass diagnostic fragment ions. Within recent years, orbitrap technology has started to replace the LTQ-FT. Originally designed for small molecule identification and quantitation, this technology has a lot of advantages in store for lipidomic applications, especially when hyphenated with the fragmentation power of linear ion trap or quadrupole technology. Although resolving power and mass accuracy are less than the LTQ-FT, they are still sufficient to provide unambiguous elemental compositions for most applications. Mass accuracy can even be increased into the sub ppm range by use of constant background signals as internal lock mass calibrants. The addition of an HCD quadrupole alleviates the low mass cutoff limitations of the LTQ and provides high resolution MS/MS spectra, although at a much slower speed than for example a quadrupole-TOF analyzer. Another advantage of the most recent LTQ-Orbitrap models in comparison to the LTQ-FT is the faster scan speed of the linear ion trap at uncompromised sensitivity, allowing acquisition of up to 20 low resolution MS/MS spectra per cycle. The combination of LTQ-Orbitrap with reversed phase HPLC was successfully used by the group of Taguchi for the determination of glycerophospholipids and PIP, either by full scan of molecular ions BIB003 or by a combination of high resolution precursor full scans and low resolution product ion scans BIB003  . Sato et al. even expanded this platform by separation of glycerophospholipids into three fractions with solid phase extraction (SPE) prior to LC-MS analysis BIB007 . Eicosanoids are an analytical challenge because they comprise a plethora of isomeric compounds. An excellent approach by reversed phase HPLC and LTQ-Orbitrap was shown by the group of Volmer BIB008 , whereby retention time, exact mass and MS/MS fragment intensities were all taken into account for successful identification of various isomers. A faster but somewhat less specific variant of this experimental setup is shown by the same group on an Orbitrap Exactive instrument coupled to reversed phase HPLC BIB006 . Although this instrumentation lacks any MS/MS capabilities, sub-ppm mass accuracies and retention time are sufficient for identification of certain glycerophospholipid classes in fast survey scans.