Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Mathias Haag, Dipl.-Ing. (FH) Born: 25 May 1981 in Darmstadt Oral examination: 17 December 2010 Development of Mass Spectrometric Methods for the Quantification of Membrane Lipids – Studies on Mitochondria, T Cells, Golgi Membranes and COPI Vesicles Referees: Prof. Dr. Felix Wieland PD. Dr. Britta Brügger 2 List of publications Christof Osman, Mathias Haag , Felix T. Wieland, Britta Brügger and Thomas Langer “A mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase Gep4“ EMBO J. 2010. Jun 16; 29(12); 1976-1987. Christof Osman, Mathias Haag , Christoph Potting, Jonathan Rodenfels, Phat Vinh Dip, Felix T. Wieland, Britta Brügger, Benedikt Westermann and Thomas Langer “The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria” J. Cell Biol. 2009. Feb 23; 184(4); 583-596. 3 Table of contents Abbreviations .................................................................................................................. 7 Abstract ........................................................................................................................... 9 Zusammenfassung ........................................................................................................10 List of figures .................................................................................................................11 List of tables...................................................................................................................12 1 Introduction ..................................................................................................13 1.1 Biological membranes .................................................................................13 1.2 Structure, synthesis and function of membrane lipids .............................13 1.2.1 Glycerophospholipids .....................................................................................13 1.2.1.1 Cardiolipin (CL) ..............................................................................................16 1.2.1.2 Phosphoinositides (PIPs) ...............................................................................17 1.2.2 Glycerolipids ..................................................................................................18 1.2.2.1 Diacylglycerol (DAG) ......................................................................................18 1.2.3 Sphingolipids ..................................................................................................19 1.2.4 Sterol lipids ....................................................................................................20 1.3 Heterogeneity of lipid membranes ..............................................................21 1.4 Lipid transport mechanisms .......................................................................22 1.5 Nano-ESI-MS/MS for lipid quantification ....................................................23 1.5.1 Nano-ESI-MS/MS for quantification of CL, DAG and PIPs .............................24 1.6 Aims of the thesis ........................................................................................25 2 Results ..........................................................................................................27 2.1 Method development ...................................................................................27 2.1.1 Quantification of CL ........................................................................................27 2.1.1.1 Mass spectrometric characterization of CL molecular species ........................27 2.1.1.2 CL quantification in the presence of mitochondrial lipid extracts .....................30 2.1.1.3 Identification and quantification of CL in yeast mitochondria...........................31 2.1.2 Quantification of DAG.....................................................................................35 2.1.2.1 Mass spectrometric characterization of DAG molecular species ....................35 2.1.2.2 Identification and quantification of DAG by MPIS ...........................................37 2.1.2.3 Identification and quantification of DAG in Golgi membranes .........................40 2.1.3 Quantification of PIP and PIP 2 ........................................................................43 2.1.3.1 Extraction behavior of endogenous PIP and PIP 2 in HeLa cells ......................43 2.1.3.2 Mass spectrometric characterization of PIP and PIP 2 molecular species ........44 4 2.1.3.3 Identification and quantification of PIP and PIP 2 by neutral loss scanning ......46 2.1.3.4 Identification and quantification of PIP and PIP 2 in HeLa cells ........................48 2.2 Method application.......................................................................................51 2.2.1 Genetic interactors of prohibitins regulate mitochondrial PE and CL ..............51 2.2.1.1 Gep1 and Ups1 regulate the level of mitochondrial PE and CL ......................51 2.2.1.2 CL and PE profiles of mitochondria lacking GEP genes .................................53 2.2.1.3 A role for Gep4 in the biosynthesis of CL .......................................................54 2.2.1.3.1 PGP accumulates in ∆gep4 mitochondria .......................................................55 2.2.1.3.2 Gep4 dephosphorylates PGP in vitro .............................................................57 2.2.2 Ilimaquinone affects DAG level in Golgi membranes ......................................58 2.2.3 TCR stimulation affects PIP, PIP 2 and DAG levels in human T cells...............60 2.3 Quantitative lipid analysis of Golgi membranes and COPI vesicles.........62 2.3.1 Characterization of subcellular fractions and generation of COPI vesicles .....62 2.3.2 Quantitative lipid analysis of subcellular fractions ...........................................63 2.3.3 Distribution of lipid species in Golgi membranes and COPI vesicles ..............64 3 Discussion ....................................................................................................71 3.1 Method development ...................................................................................71 3.1.1 Quantification of CL ........................................................................................71 3.1.2 Quantification of DAG.....................................................................................72 3.1.3 Quantification of PIP and PIP 2 ........................................................................73 3.2 Method application.......................................................................................75 3.2.1 Genetic interactors of prohibitins regulate mitochondrial PE and CL ..............75 3.2.2 Ilimaquinone affects DAG level in Golgi membranes ......................................78 3.2.3 TCR stimulation affects PIP, PIP 2 and DAG levels in human T cells...............79 3.3 Quantitative lipid analysis of Golgi membranes and COPI vesicles.........80 3.3.1 Characterization of subcellular fractions and generation of COPI vesicles .....80 3.3.2 Quantitative lipid analysis of subcellular fractions ...........................................80 3.3.3 Distribution of lipid species in Golgi membranes and COPI vesicles ..............81 3.3.4 Mechanisms of segregation............................................................................82 4 Materials and methods ................................................................................84 4.1 Materials .......................................................................................................84 4.1.1 Chemicals and materials ................................................................................84 4.1.2 Lipids .............................................................................................................85 4.1.3 Instruments ....................................................................................................85 5 4.2 Methods ........................................................................................................85 3 4.2.1 Cell culture and myo -[ H]-inositol labeling ......................................................85 4.2.2 Phosphorus assay (micro determination) .......................................................86 4.2.3 Preparation of lipid standards .........................................................................86 4.2.4 Preparation of primary human T cells and TCR stimulation ............................86 4.2.5 Purification of Golgi membranes and COPI vesicles ......................................87 4.2.6 Lipid extraction procedures ............................................................................87 4.2.6.1 Lipid extraction from GEP mitochondria for PE and CL analysis ....................87 4.2.6.2 Lipid extraction from wt, ∆gep4 and ∆crd1 mitochondria ................................88 3 4.2.6.3 Phosphoinositide extraction from myo -[ H]-inositol labeled HeLa cells ...........89 4.2.6.4 Phosphoinositide extraction from HeLa cells ..................................................90 4.2.6.5 Lipid extraction from HeLa Golgi membranes for DAG quantification .............90 4.2.6.6 Lipid extraction from NRK Golgi membranes for DAG quantification ..............90 4.2.6.7 Lipid extraction