Z3 - Proteomics and Lipidomics Investigations by Mass Spectrometry and NMR

The functionality of adipose tissue is characterized by different molecular processes. This includes (1) the characteristic metabolism of the tissue and the related cells, (2) the interactions with the systemic metabolism by metabolites as well as adipokines and (3) the interactions with immune cells such as macrophages. The detailed analysis of these processes is challenging and requires different bioanalytical techniques.

In particular, we will develop assays for the determination of serum and tissue proteins with the focus on adipokines such as chemerin and leptin. The application of these newly developed "high-throughput" assays will enable the correlation between gene variations and phenotype, on the one hand, and the adipokine concentration, on the other hand. By using global proteomics we will characterize the changes in the differentiation of adipocytes and the related adipose tissues. Using phosphoproteomics we will also be able to identify the relevant signaling pathways and to quantify the related metabolites. Finally, the determination of obesity-specific serum and tissue will help to characterize the impact of altered obese tissue functions.

In addition to protein analysis, the analysis of lipids plays also an important role in this project. Beside the investigation of "storage lipids" (i.e. triacylglycerols) in dependence on the diet composition and selected metabolic disorders which will be induced by the "knockout" of the related enzymes, the analysis of phospholipids will be also of major relevance. We are particularly interested in phospholipids with messenger functions and oxidized lipids which are generated by reactive oxygen species (ROS) at inflammatory conditions. In addition to mass spectrometric techniques (MALDI and ESI) NMR methods will be also applied: although NMR is not very sensitive, this technique offers some important advantages. Using 31P NMR the analysis of virtually all phospholipid classes in complex mixtures is possible and there is no need of previous chromatographic separation. Finally, high resolution 13C-NMR enables the identification of the positions of the different fatty acyl residues in triglycerols.

Figure 1: SRM method which enables the selective determination of adipozytokins. (A) This method uses two different mass filters and quantitative data are obtained by characteristic "mass transitions". (B) Oberbach et al. (2015) provided evidence that this method can be used to quantify at least 4 different serum proteins.

Figure 2: Impact of the supplied diet on the fatty acyl composition of brown adipose tissue. (A) The characteristic fatty acid compositions of the diet and the adipose tissue show pronounced differences. (B) By using 13C NMR the positions of the individual fatty acyl residues can be determined. In (2A) the impact of different diets on the fatty acyl composition of brown adipose tissue (from mice) is shown. In the used x:y nomenclature "x" denotes the number of carbon atoms in the fatty acid while "y" denotes the number of double bonds. The determination of the positions of the different acyl residues can be achieved by 13C NMR spectroscopy (2B). In the majority of cases sn-1,3- and sn-2 positions can be differentiated.

Mandić AD, Woting A, Jaenicke T, Sander A, Sabrowski W, Rolle-Kampcyk U, von Bergen M, Blaut M. Clostridium ramosum regulates enterochromaffin cell development and serotonin release. Sci Rep. 2019 Feb 4;9(1):1177.

Ditz T, Schnapka-Hille L, Noack N, Dorow J, Ceglarek U, Niederwieser D, Schiller J, Fuchs B, Cross M. Phospholipase A2 products predict the hematopoietic support capacity of horse serum. Differentiation. 2018 Dec 6;105:27-32. 

Leopold J, Popkova Y, Engel KM, Schiller J. Recent Developments of Useful MALDI Matrices for the Mass Spectrometric Characterization of Lipids. Biomolecules. 2018 Dec 13;8(4). 

Vorselen D, van Dommelen SM, Sorkin R, Piontek MC, Schiller J, Döpp ST, Kooijmans SAA, van Oirschot BA, Versluijs BA, Bierings MB, van Wijk R, Schiffelers RM, Wuite GJL, Roos WH.The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis. Nat Commun. 2018 Nov 23;9(1):4960.

Leopold J, Popkova Y, Engel KM, Schiller J. Visualizing phosphatidylcholine via mass spectrometry imaging: relevance to human health. Expert Rev Proteomics. 2018 Sep 21.

Kratochvil I, Hofmann T, Rother S, Schlichting R, Moretti R, Scharnweber D, Hintze V, Escher BI, Meiler J, Kalkhof S, von Bergen M. MEHP and MEOHP but not DEHP bind productively to the peroxisome proliferator-activated receptor γ. Rapid Commun Mass Spectrom. 2018 Aug 7.

Engel K, Griesinger H, Schulz M, Schiller J.Normal Phase versus Reversed Phase TLC to monitor Oxidized Phosphatidylcholines by TLC/Mass Spectrometry. Rapid Commun Mass Spectrom. 2018 Jul 18.

Schröter J, Fülöp A, Hopf C, Schiller J. The combination of 2,5-dihydroxybenzoic acid and 2,5-dihydroxyacetophenone matrices for unequivocal assignment of phosphatidylethanolamine species in complex mixtures. Anal Bioanal Chem. 2018 Mar;410(9):2437-2447.

Herbert D, Franz S, Popkova Y, Anderegg U, Schiller J, Schwede K, Lorz A, Simon JC, Saalbach AHigh fat diet exacerbates early psoriatic skin inflammation independent of obesity: Saturated fatty acids as key players. J Invest Dermatol. 2018 Mar 29. pii: S0022-202X(18)31858-X.

Junge KM, Leppert B, Jahreis S, Wissenbach DK, Feltens R, Grützmann K, Thürmann L, Bauer T, Ishaque N, Schick M, Bewerunge-Hudler M, Röder S, Bauer M, Schulz A, Borte M, Landgraf K, Körner A, Kiess W, von Bergen M, Stangl GI, Trump S, Eils R, Polte T, Lehmann I. MEST mediates the impact of prenatal bisphenol A exposure on long-term body weight development. Clin Epigenetics. 2018 Apr 20;10:58. 

Engel KM, Schiller J, Müller K, Dannenberger D, Jakop U. The phospholipid composition of kangaroo spermatozoa verified by mass spectrometric lipid analysis. Lipids. 2017;52:857-69.

Engel KM, Schiller J. A comparison of PC oxidation products as detected by MALDI-TOF and ESI-IT mass spectrometry. Chem Phys Lipids. 2017;203:33-45.

Popkova Y, Schiller J. TAG suppression by CsCl addition. Rapid Commun Mass Spectrom. 2017;31:411-8.

Schröter J, Süß R, Schiller J. MALDI-TOF MS to monitor the kinetics of phospholipase A2-digestion of oxidized phospholipids. Methods. 2016;104:41-7.

Stelzner K, Herbert D, Popkova Y, Lorz A, Schiller J, Gericke M, Klöting N, Blüher M, Franz S, Simon JC, Saalbach A. Free fatty acids sensitize dendritic cells to amplify TH1/TH17-immune responses. Eur J Immunol. 2016;46:2043-53.

Kühn T, Floegel A, Sookthai D, Johnson T, Rolle-Kampczyk U, Otto W, von Bergen M, Boeing H, Kaaks R. Higher plasma levels of lysophosphatidylcholine 18:0 are related to a lower risk of common cancers in a prospective metabolomics study. BMC Med. 2016;14:13.

Popkova Y, Meusel A, Breitfeld J, Schleinitz D, Hirrlinger J, Dannenberger D, Kovacs P, Schiller J. Nutrition-dependent changes of adipose tissue compositions monitored by NMR, MS and chromatographic methods. Anal Bioanal Chem. 2015;407:5123-33.

Klöting N, Hesselbarth N, Gericke M, Kunath A, Biemann R, Chakaroun R, Kosacka J, Kovacs P, Kern M, Stumvoll M, Fischer B, Rolle-Kampczyk U, Feltens R, Otto W, Wissenbach DK, von Bergen M, Blüher M. Di-(2-ethylhexyl)-phthalate (DEHP) causes impaired adipocyte function and alters serum metabolites. PLoS One. 2015;10:e0143190.

Herberth G, Offenberg K, Rolle-Kampczyk U, Bauer M, Otto W, Röder S, Grützmann K, Sack U, Simon JC, Borte M, von Bergen M, Lehmann I; LINA Study Group. Endogenous metabolites and inflammasome activity in early childhood and links to respiratory diseases. J Allergy Clin Immunol. 2015;136:495-7.

Fuchs B, Popkova Y, Süß R, Schiller J. Separation of (Phospho)lipids by thin-layer chromatography. In: Poole C. (Ed.): "Handbooks in Separation Science: Instrumental Thin-Layer Chromatography". Elsevier, Amsterdam, 2015, pp. 375-405. ISBN 978-0-12-417223-4 .

Schiller J, Fuchs B, Süß R, Popkova Y, Griesinger H, Matheis K, Minarik S, Oberle M, Schulz M. TLC - MALDI MS for the Analysis of Lipids. "Planar Chromatography – Mass Spectrometry", Taylor and Francis Verlag, 2015, pp. 213-232, ISBN 9781498705882 .

Eibisch M, Popkova Y, Süß R, Schiller J, Dannenberger D. Evaluation of a commercial enzymatic test kit regarding the quantitative analysis of different free fatty acids. Anal Bioanal Chem. 2014;406:7401-5.

Pirkl A, Meier M, Popkova Y, Letzel M, Schiller J, Schnapp A, Dreisewerd K. Analysis of free fatty acids by ultraviolet laser desorption ionization mass spectrometry from using insect wings as hydrophobic sample substrates. Anal Chem. 2014;86:10763-71.

Griesinger H, Fuchs B, Süß R, Matheis K, Schulz M, Schiller J. The thickness of the stationary phase determines the quality of TLC / MALDI mass spectra of lipids. Anal Biochem. 2014;451:45-7.

Oberbach A, Schlichting N, Neuhaus J, Kullnick Y, Lehmann S, Heinrich M, Dietrich A, Mohr FW, von Bergen M, Baumann S. Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation. J Proteome Res. 2014;13:5784-800.

Oberbach A, Blüher M, Wirth H, Till H, Kovacs P, Kullnick Y, Schlichting N, Tomm JM, Rolle-Kampczyk U, Murugaiyan J, Binder H, Dietrich A, von Bergen M. Combined proteomic and metabolomic profiling of serum reveals association of the complement system with obesity and identifies novel markers of body fat mass changes. Proteome Res. 2011;10:4769-88


Yulia Popkova, Postdoc

Alexandra Schaffert, Phd student

Laura Krieg, Phd student