Metabolomics and Lipidomics Profiling of a Combined Mitochondrial

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Metabolomics and lipidomics profiling of a combined mitochondrial plus endoplasmic reticulum fraction of human fibroblasts: a robust tool for clinical studies Charlotte Veyrat-Durebex, Cinzia Bocca, Stéphanie Chupin, Judith Kouassi Nzoughet, Gilles Simard, Guy Lenaers, Pascal Reynier, and Hélène Blasco J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00637 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 8, 2017

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Journal of Proteome Research

Metabolomics and lipidomics profiling of a combined mitochondrial plus endoplasmic reticulum fraction of human fibroblasts: a robust tool for clinical studies

Charlotte Veyrat-Durebex1,2*, Cinzia Bocca2, Stéphanie Chupin1, Judith Kouassi NZoughet2, Gilles Simard1, Guy Lenaers2, Pascal Reynier1,2, Hélène Blasco2,3,4

1

Département de Biochimie et Génétique, Centre Hospitalier Universitaire, Angers, France

2

Equipe Mitolab, Institut MITOVASC, UMR CNRS 6015, INSERM 1083, Université

d’Angers, France 3

Université François-Rabelais, INSERM U930, Tours, France

4

Laboratoire de Biochimie et Biologie moléculaire, CHRU de Tours, France

* Correspondence to: Dr Charlotte VEYRAT-DUREBEX Département de Biochimie et Génétique, CHU d’Angers, 4 rue Larrey, 49933 ANGERS Cedex 9 Tel +33 (0)2 41 35 32 28 [email protected]

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ACS Paragon Plus Environment


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Abstract Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles functions, dysfunctions and interactions. Here, we developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with an excellent reproducibility. Seventy two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12 % among which many specific of the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and “omics” combination will be useful for investigating the pathophysiology of complex diseases.

Keys words: metabolomics, lipidomics, mitochondria, endoplasmic reticulum

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Introduction Mitochondria are the site of the energy production and are also involved in most biosynthesis pathways, in cell signaling, calcium buffering, reactive oxygen species (ROS) metabolism and apoptosis 1. Endoplasmic reticulum (ER) is mainly involved in protein and lipid biosynthesis, sequesters calcium, and transfers it to mitochondria upon stimulation. Mitochondria and ER are organelles physically and functionally coupled: i) ER contributes to the mitochondrial biogenesis by providing phospholipids and proteins 2; ii) they are both involved in calcium-related cell signaling and in cell stress response to protein unfolding

3-5

;

iii) they are physically closely related by the mitochondria-associated membranes (MAM) that constitute the sites of lipids, calcium and proteins transfer from the ER towards mitochondria, with a crucial regulatory role in cell homeostasis

6-8

role in the mitochondrial fission, apoptosis and ROS generation

6, 8, 9

. MAM also display a key . Consequently, we may

suggest that ER-mitochondria miscommunication could participate to various diseases. It seems interesting to explore the mitochondria-MAM-ER system as a whole.

Mitochondrial dysfunctions are usually investigated through the oxidative phosphorylation (OXPHOS), apoptosis, mitochondrial DNA maintenance, network dynamics, ROS production and calcium fluxes. ER stress is explored via the 3 pathways of unfolded protein response (UPR) activation, the calcium fluxes and the structure of the organelle by cell imaging. Patients’ fibroblasts remain models commonly used in diagnosis for these explorations. Although standardized and informative, these explorations remained limited since they focused on well- and already-known pathophysiological concepts. As these explorations cover only some definite organelles’ functions, they remain not informative enough for 3



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complex disease description. The close partnership between mitochondria and the ER, places these organelles at the core of many pathophysiological mechanisms, thus being increasingly studied 8.

Metabolomics and lipidomics have emerged as analytical-statistical powerful approaches for the detection of subtle novel pathophysiological mechanisms, due to their ability to explore in depth many metabolic pathways. Recently, the investigation by targeted metabolomics of the fibroblasts of patients affected by a mitochondrial inherited optic neuropathy with an OXPHOS defect, revealed an unexpected UPRER activation pharmacologically reversible, opening a new therapeutic strategy

10

. Recent studies reported mitochondria’ lipidome

11-14

,

but the long procedures used to isolate mitochondria are incompatible with metabolomics analyses due to the risk of metabolites degradation 15. We suggest that it would be interesting to develop new faster isolation procedures of mitochondria, allowing analysis of both metabolome and lipidome.

Herein, we report the development and validation of a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts, useful for combined metabolomics and lipidomics analyses by mass spectrometry.

Experimental Section Study methodology This is a 3-steps study; we first evaluated 4 procedures of mitochondria-ER purification, from which we selected the one providing the highest purity and yield of mitochondria, as assessed by Western blot. Secondly, the extracts were analyzed by high resolution mass spectrometry 4


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(HRMS)-based metabolomics and lipidomics techniques. The reproducibility was verified using ten independent replicates. Finally, the number of compounds detected with low variability was assessed by both targeted and untargeted metabolomics and lipidomics strategies.

Purification of mitochondria-ER enriched fraction Cell cultures Human fibroblasts were cultured in a medium consisting of two-thirds Dulbecco’s modified Eagle medium with nutrient mixture F12 (DMEM-F12, PAN Biotech), one-third AmnioMAX™ (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (PAN Biotech) at 37°C and 5% CO2. To avoid artefacts due to senescence, all experiments were conducted on fibroblast cultures between the 6th and the 25th passages

16

. Cells were harvested using

trypsin, and cell pellets containing 10 million cells were stored at -80°C until extraction.

Mitochondria-ER enriched fraction purification procedures Cell pellets were resuspended in 200 µL of cold NaCl 0.9% solution. We evaluated 4 different purification procedures of mitochondria-ER based on different cell lysis procedures: 1) 200 µL of digitonin solution (4 mg/mL) were added to cell suspension and incubated 10 min at 4°C; 2) cell suspension was transferred to a 0.5 mL homogenizer tube prefilled with 1.4 mm ceramic beads (Soft tissue homogenizing CK14, Bertin Instruments, Montigny-leBretonneux) and cell lysis was achieved in a Precellys24® homogenizer (Bertin Instruments) using 2 cycles of grinding 10 s at 2500 rpm followed by 10 s at 4000 rpm at 4°C; 3) same procedure as 2) but with one cycle of grinding 10 s at 2500 rpm at 4°C; 4) same procedure as 2) but with one cycle of grinding 5 s at 2500 rpm at 4°C. 5



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Samples resulting from each cell lysis procedure were then transferred in a polypropylene microtube and centrifuged at 1000 g for 10 min at 4°C. The pellet corresponding to the nuclear fraction and cellular debris was kept for Western blot analysis, and the resulting supernatant was transferred to another microtube to further perform mitochondria-ER purification. This was achieved by a centrifugation at 10 000 g for 10 min at 4°C. The resulting pellet was kept as the mitochondria-ER fraction and the supernatant as the cytosolic fraction. The figure 1 outlines the general methodology of the 4 methods.

Figure 1

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Western blot analysis Nuclear and mitochondria-ER fractions were resuspended in water containing anti-proteases (Protease inhibitor cocktail, #88666, Life Technologies). Proteins were quantified in the different fractions using BC Assay procedure (Interchim). Proteins (40 µg) were separated by SDS-PAGE in 10 or 12.5% poly-acrylamide gels and transferred on polyvinylidene difluoride membranes using Trans-Blot® Turbo™ transfer system (Biorad). Blots were probed overnight at 4°C with anti-tubulin, voltage-dependent anion channel (VDAC), cytochrome c, calnexin and the nuclear matrix protein p84 antibodies (Abcam, #ab7291, #ab14734, #ab110325 and #ab133615, Euromedex antibody anti-p84 #GTX70220), diluted to 1/1000 in Tris-buffer solution containing 0.1% Tween 20 (TTBS) with 1 % of skimmed milk. The membranes were then washed at least three times in TTBS and incubated for 1h at room temperature with the appropriate secondary antibody (VWR, polyclonal donkey anti-rabbit or sheep anti-mouse antibody diluted to 1/10 000, #NA931 and #NA934). The protein-antibody complexes were detected using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) according to the manufacturer protocol and analyzed by Odyssey®Fc Imaging System (LI-COR Biosciences).

Metabolomics and lipidomics analyses Metabolites and lipids extraction protocols are described in additional methods (Supporting Information). HRMS system was based on UPLC Ultimate 3000 system (Dionex) coupled to a Q-Exactive Mass Spectrometer (ThermoFisher Scientific, Bremen, Germany) using electrospray ionization (positive and negative Electrospray Ionization (ESI+ and ESI-)). Analytical systems used for metabolomics and lipidomics analyses are described in additional methods (Supporting Information). 7



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Data analysis XCMS online (https://xcmsonline.scripps.edu/) was used to perform untargeted metabolomics and lipidomics analyses, and provided multiple features corresponding to ions detected, using specific parameters of Q-Exactive technology 17. For metabolomics targeted data analysis, TraceFinder 4.1 software (Thermo Fisher Scientific) was used for metabolites identification and peak integration using an in-house library 18. For lipidomics targeted data analysis, LipidSearch™ was employed for lipids identification using MS and MS² spectra, and TraceFinder 4.1 software for data processing. The level of compound identification was done according to Sumner et al. 19 Compounds’ levels were normalized to the sum of all measured compounds, and compounds exhibiting more than 35% of coefficient of variation (CV) in the 10 biological replicate samples were excluded in the final dataset summarizing metabolomics and lipidomics signatures. In addition, unsupervised principal components analysis (PCA)

20

was performed

to identify similarities or differences between the metabolomics and lipidomics profiles of the 10 replicate samples (SIMCA® P+ v14.1 software, Umetrics, Umea, Sweden). Score and DModX plots were visually inspected for grouping, trends and outliers in the data

21

. Venn

diagrams were drawn using Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/) to highlight common and distinct compounds between the different ionization modes.

Results and Discussion Selection of the optimized procedure for mitochondria-ER purification Western blot analyses of the mitochondria-ER, nuclear and cytosolic fractions obtained from the 4 purification methods are shown in the figure 2, and revealed that method 4 (M4) provided the highest yield of mitochondria in the mitochondria-ER fraction, with the highest 8


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levels of cytochrome c (mitochondrial matrix) and VDAC (mitochondrial outer membrane), and the lowest contamination by nuclear (p84) and cytosolic (tubulin) markers. The mitochondria-ER fraction contained the majority of ER fraction in the four methods tested, as revealed by the calnexin staining. This result is consistent with studies reported that the fraction of ER cosedimenting with mitochondria contains lipid biosynthesis enzymes and is mainly composed of MAM 22. We also found that method M4 provided nuclear and cytosolic fractions with the lowest level of the VDAC protein, highlighting a minimal loss of intact mitochondria in these fractions. These findings suggested that majority of mitochondria isolated with method M4 are intact and in a quantity suitable for metabolomics approaches. The preservation of intact mitochondria allows us to investigate its content and metabolic status. Thus, using the M4 protocol, we obtained a sub-cellular fraction containing the majority of ER and mitochondria.

Figure 2

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Large coverage of metabolites Targeted data analysis of metabolomics acquisitions in ESI- and ESI+ enabled the detection of 46 and 49 metabolites, respectively, having a CV

Metabolomics and Lipidomics Profiling of a Combined Mitochondrial

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