Kai Schuhmann

A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language

Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the molecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes.

Top-down shotgun lipidomics relies on direct infusion of total lipid extracts into a high-resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top-down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high-resolution spectra in both polarity modes with sub-ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle.

LipidXplorer: A Software for Consensual Cross-Platform Lipidomics

LipidXplorer is the open source software that supports the quantitative characterization of complex lipidomes by interpreting large datasets of shotgun mass spectra. LipidXplorer processes spectra acquired on any type of tandem mass spectrometers; it identifies and quantifies molecular species of any ionizable lipid class by considering any known or assumed molecular fragmentation pathway independently of any resource of reference mass spectra. It also supports any shotgun profiling routine, from high throughput top-down screening for molecular diagnostic and biomarker discovery to the targeted absolute quantification of low abundant lipid species. Full documentation on installation and operation of LipidXplorer, including tutorial, collection of spectra interpretation scripts, FAQ and user forum are available through the wiki site at: https://wiki.mpi-cbg.de/wiki/lipidx/index.php/Main_Page.

Shotgun lipidomics on high resolution mass spectrometers.

Despite their compositional complexity, lipidomes comprise a large number of isobaric species that cannot be distinguished by conventional low resolution mass spectrometry and therefore in-depth MS/MS analysis was required for their accurate quantification. Here we argue that the progress in high resolution mass spectrometry is changing the concept of lipidome characterization. Because exact masses of isobaric species belonging to different lipid classes are not necessarily identical, they can now be distinguished and directly quantified in total lipid extracts. By streamlining and simplifying the molecular characterization of lipidomes, high resolution mass spectrometry has developed into a generic tool for cell biology and molecular medicine.

Bottom-up shotgun lipidomics by higher energy collisional dissociation on LTQ Orbitrap mass spectrometers.

Higher energy collision dissociation (HCD) is a complementary fragmentation tool that has recently become available on mass spectrometers of the LTQ Orbitrap family. We report on a shotgun bottom-up lipidomics approach that relies on HCD of the isolated lipid precursors. HCD, together with the high mass resolution and mass accuracy of the Orbitrap analyzer, improved the confidence of molecular species assignment and accuracy of their quantification in total lipid extracts. These capabilities were particularly important for accounting for biologically interesting lipid species comprising polyunsaturated and odd numbered fatty acid moieties. We argue that now both bottom-up and top-down shotgun lipidomics could be performed on the same instrumentation platform.

Maradolipids: Diacyltrehalose Glycolipids Specific to Dauer Larva in Caenorhabditis elegans

In response to harsh environmental conditions, such as overcrowding or starvation, the nematode C. elegans interrupts and arrests its reproductive life cycle by forming a specific dauer (enduring) larva. Dauer larvae have very distinct metabolism, morphology, and enhanced stress resistance for surviving unfavorable environmental conditions. They express high amounts of stress-protective proteins such as the heat-shock protein Hsp90, superoxide dismutase, and catalase . They also remodel their body surfaces—they build a dauer-specific cuticle and seal the pharynx with a cuticular block . Although, the dauer larva formation pathway is biologically well investigated, there is not much information about the chemical means by which dauer larvae resist the various kinds of environmental stresses. Similar to some other organisms, lipids might play an important role in the adaptation process of dauer larvae to harsh conditions. We asked whether the transition from reproductive stages to the dauer larva is associated with global changes in the lipid composition or metabolism. For this purpose we used a temperature-sensitive mutant of daf-2(e1370) that reproduces at 15 8C or 20 8C but forms dauer larvae at 25 8C . Lipids were extracted from daf-2(e1370) worms grown at 20 8C and 25 8C (Figures 1 a and b, respectively) and separated by two-dimensional (2D) thin-layer chromatography (TLC) that resolved the major lipid classes: glycerophospholipids, ceramides, glycosphingolipids, fatty acids, sterols, etc. The plates were sprayed with the Molisch reagent, which specifically stains carbohydrate-containing lipids in purple and all other lipid classes in yellow-brown on the same TLC plates. As seen, there is a significant difference between reproductive L3 larvae and dauer larvae (compare Figures 1a and b): In addition to two Molisch-positive (purple) forms of glucosylceramides (GlucCer), a spot that is visible exclusively on the TLC containing the dauer larvae is observed (arrowhead). This spot appeared to be specific to dauer larva; that is, it could not be detected either in the mixed population of wildtype worms grown at 20 8C and 25 8C, or in any other individual reproductive larval stages (L1 to L4, adults; not shown). Most importantly, dauer larvae obtained from starved plates of wild-type worms (N2) displayed a spot of comparable strength (see Figure 1 a (arrowhead) in the Supporting Information). Hence we conclude that the spot represents a genuine lipid component of the natural dauer larvae, which does not depend on the genetic background or temperature. Mobility of the dauer-larva-specific lipid on TLC and its positive reaction to the Molisch reagent suggested that it might be a dauer-specific glycosphingolipid. To test this possibility, we isolated neutral glycolipids (NGL) from dauer larvae (see Figure 2a in the Supporting Information). The dauer-larva-specific spot was indeed found in the glycolipid fraction (NGL, arrowhead). In contrast to glycosphingolipids, however, this lipid was susceptible to saponification (see Figure 1b in the Supporting Information; compare patterns before and after saponification—the spot indicated by arrowhead is absent after the saponification). Our observation indicates that this lipid does not belong to the class of glycosphingolipids and must contain at least one ester bond (amide bonds of glycosphingolipids cannot be cleaved by saponification). On the basis of its occurrence exclusively in dauer larvae and its chemical dissimilarity to glycosphingolipids, we call this lipid maradolipid (from maradi, enduring/ dauer in Georgian). [*] S. Penkov, F. Mende, V. Zagoriy, C. Erkut, K. Schuhmann, Dr. D. Schwudke, J. M ntler, Dr. A. Shevchenko, Prof. Dr. T. V. Kurzchalia Max Planck Institute for Molecular Cell Biology and Genetics Pfotenhauerstraße 108, 01307 Dresden (Germany) Fax: (+ 49)351-210-2000 E-mail: kurzchalia@mpi-cbg.de

Gender, Contraceptives and Individual Metabolic Predisposition Shape a Healthy Plasma Lipidome.

Lipidomics of human blood plasma is an emerging biomarker discovery approach that compares lipid profiles under pathological and physiologically normal conditions, but how a healthy lipidome varies within the population is poorly understood. By quantifying 281 molecular species from 27 major lipid classes in the plasma of 71 healthy young Caucasians whose 35 clinical blood test and anthropometric indices matched the medical norm, we provided a comprehensive, expandable and clinically relevant resource of reference molar concentrations of individual lipids. We established that gender is a major lipidomic factor, whose impact is strongly enhanced by hormonal contraceptives and mediated by sex hormone-binding globulin. In lipidomics epidemiological studies should avoid mixed-gender cohorts and females taking hormonal contraceptives should be considered as a separate sub-cohort. Within a gender-restricted cohort lipidomics revealed a compositional signature that indicates the predisposition towards an early development of metabolic syndrome in ca. 25% of healthy male individuals suggesting a healthy plasma lipidome as resource for early biomarker discovery.

Lipidomic profiling before and after Roux-en-Y gastric bypass in obese patients with diabetes

Bariatric surgery is a well-established approach to improve metabolic disease in morbidly obese patients with high cardiovascular risk. The post-operative normalization of lipid metabolism has a central role in the prevention of future cardiovascular events. The aim of the present study therefore was to characterize changes of plasma lipidomic patterns, consisting of 229 lipid species of 13 lipid classes, 3 months after Roux-en-Y gastric bypass (RYGB) in morbidly obese patients with and without diabetes. RYGB resulted in a 15–32% decrease of body mass index, which was associated with a significant reduction of total cholesterol (TC, −28.3%; P=0.02), LDL-cholesterol (LDL-C, −26.8%; P=0.03) and triglycerides (TGs, −63.0%; P=0.05) measured by routine clinical chemistry. HDL-cholesterol remained unchanged. The effect of RYGB on the plasma lipidomic profile was characterized by significant decreases of 87 lipid species from triacylglycerides (TAGs), cholesterol esters (CholEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), phosphatidylethanolamine ethers (PEOs), phosphatidylinositols (PIs) and ceramides (Cers). The total of plasma lipid components exhibited a substantial decline of 32.6% and 66 lipid species showed a decrease by over 50%. A direct correlation with HbA1C values could be demonstrated for 24 individual lipid species (10 TAG, three CholE, two LPC, one lysophosphatidylcholine ethers (LPCO) (LPC ether), one PC, two phosphatidylcholine ethers (PCO) and five Cer). Notably, two lipid species (TAG 58:5 and PEO 40:5) were inversely correlated with HbA1C. LPCO, as single whole lipid class, was directly related to HbA1C. These data indicate that RYGB-induced modulation of lipidomic profiles provides important information about post-operative metabolic adaptations and might substantially contribute to improvements of glycemic control. These striking changes in the human plasma lipidome may explain acute, weight independent and long-term effects of RYGB on the cardiovascular system, mental status and immune regulation.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950–Metabolites in Frozen Human Plasma

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950–Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Altered Desaturation and Elongation of Fatty Acids in Hormone-Sensitive Lipase Null Mice

Background Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored lipids, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. The aim of this study was to define lipid profiles in plasma, white adipose tissue (WAT) and liver of HSL null mice, in order to better understand the role of this multifunctional enzyme. Methodology/Principal Findings This study used global and targeted lipidomics and expression profiling to reveal changed lipid profiles in WAT, liver and plasma as well as altered expression of desaturases and elongases in WAT and liver of HSL null mice on high fat diet. Decreased mRNA levels of stearoyl-CoA desaturase 1 and 2 in WAT were consistent with a lowered ratio of 16∶1n7/16∶0 and 18∶1n9/18∶0 in WAT and plasma. In WAT, increased ratio of 18∶0/16∶0 could be linked to elevated mRNA levels of the Elovl1 elongase. Conclusions This study illustrates the importance of HSL for normal lipid metabolism in response to a high fat diet. HSL deficiency greatly influences the expression of elongases and desaturases, resulting in altered lipid profiles in WAT, liver and plasma. Finally, altered proportions of palmitoleate, a recently-suggested lipokine, in tissue and plasma of HSL null mice, could be an important factor mediating and contributing to the changed lipid profile, and possibly also to the decreased insulin sensitivity seen in HSL null mice.