International Summit on Current trends in Mass Spectrometry July 13-15, 2015 New Orleans, Louisiana.

Transcription

1 International Summit on Current trends in Mass Spectrometry July 13-15, 2015 New rleans, Louisiana

2 utline Introduction Duchenne NAFLD Cystic fibrosis Fabry disease Peptidolipids Cholesterol Environmental Preservative Anti-acne drug 2

3 Introduction After dissection, fast freezing at -80 C Tissue is maintained with CT glue µm sections cut with a cryostat ( -20 C) Tissue sections are deposited directly onto a stainless steel plate, a silicon wafer, or a glass plate Dehydrated under vacuum Fixed onto the standard sample holder Blade 12 μm section 3

4 Introduction 4

5 Introduction Very good focus at unit mass resolution optical image: field of view: 700 x 700 µm² field of view: 55 x 55 µm² primary ion: Bi + 3 pixel size 215 nm cholesterol superposition superimposition total ion phosphocholine D. Touboul et al. J. Am. Soc. Mass Spectrom. 2005, 16,

6 Introduction DESI : Roger Webb (Monday morning) Spatial resolution MALDI-TF-TF TF-SIMS 50 µm 400 nm - 2 µm MALDI : Mehran F Moghaddam (Monday Morning) Dehydrated, Dehydrated MeV SIMS Sample : Roger Webb Homogeneous (Monday Afternoon) No fixation matrix coating No matrix Mass range m/z > 200 m/z 1000 Compounds Proteins, Peptides, Lipids, Drugs, Metabolites, Lipids Glycosphingolipi ds Cyclopeptides, Drugs, Metabolites, Elements, Low Energy Analyzer Cs+ SIMS TF-MS/MS : PurushottamTF-MS Chakraborty (Tuesday Morning) 6

7 Introduction DESI : Roger Webb (Monday morning) Spatial resolution MALDI-TF-TF TF-SIMS 50 µm 400 nm - 2 µm MALDI : Mehran F Moghaddam (Monday Morning) Dehydrated, Dehydrated MeV SIMS Sample : Roger Webb Homogeneous (Monday Afternoon) No fixation matrix coating No matrix Mass range m/z > 200 m/z 1000 Lipids Proteins, Glycosphingolipi Peptides, ds Lipids, 20 KeV Compounds Bi 3+ TF-SIMS Cyclopeptides, Drugs, Drugs, Metabolites, Metabolites, Elements, Low Energy Analyzer Cs+ SIMS TF-MS/MS : PurushottamTF-MS Chakraborty (Tuesday Morning) 7

8 Introduction Spatial resolution Sample MALDI-TF-TF TF-SIMS µm 400 nm - 2 µm Dehydrated, Homogeneous matrix coating Dehydrated No fixation No matrix Mass range m/z > 200 m/z 1500 Compounds Proteins, Peptides, Lipids, Drugs, Metabolites, Lipids Glycosphingolipids Cyclopeptides, Drugs, Metabolites, Elements, Analyzer TF-MS/MS TF-MS 8

9 Introduction 9

10 Introduction Fatty acids Glycerophospholipids: P H H N Phosphatidylcholine α-tocopherol (Vit E) H H H H P H X X = H, Na, K, Ca... Sphingomyelin H N P HN Phosphatidylinositol H Cholesterol Triglycerides Diglycerides H 10

11 Introduction 500 µm Microscope optical image = C30:(2-0) mc:73 tc: = C32:(3-0) mc:185 tc: Diglycerides = C34:(3-0) mc:244 tc: Sphingomyelins and cholesterol = C36:(4-0) mc:161 tc: = SM fragment mc:6 tc: = SM fragment mc:7 tc:16129 (mode -) 385 = Cholesterol mc:6 tc:14556 (mode -) 642 = SM fragment mc:7 tc:20513 (mode -) 687 = SM fragment mc:9 tc:26892 (mode -) Vitamin E (mode+) mc:7 tc:12860 Vitamin E (mode -) mc:14 tc:34882 Vitamin E 11

12 Introduction Adipocytes (infiltrates) Damaged area (necrotic tissue) Muscular fibers N. Tahallah, A. Brunelle, S. De La Porte,. Laprévote, J. Lipid Res. 2008, 49,

13 Introduction 1000 µm H&E staining (adjacent sections) 1000 µm Control liver Fatty liver Accumulation of lipids (mainly diacyl- and triacylglycerols) in hepatocytes, The predominant risk factor for NAFLD appears to be insulinresistance related to the metabolic syndrome, n stained sections, lipid vesicles are observed in fatty liver but are not observable in healthy liver, 13

14 Introduction Control liver Fatty liver - steatotic area Intensity per unit area (counts x10 3 ) MAG Vit.E Chol DAG m/z PC Strong decrease of vitamin E signal Detection of triacylglycerols Strong increase of diacylglycerols Intensity per unit area (counts x10 3 ) MAG Chol DAG m/z PC TAG D. Debois et al., Anal. Chem. 2009, 81,

15 Introduction Cholesterol µm 50 Sum of DAG ions C30 DAGs C32 DAGs C34 DAGs C36 DAGs

16 Intensité par unité de surface 3 (coups x 10 ) MAG MAG Cholestérol Introduction 20 DAG C µm Cholestérol 10 DAG C32 DAG C30 DAG C m/z Red = DAG C30 Green = DAG C36 Red = Unsaturated lipids Green = saturated lipids F. Le Naour et al. Plos ne, 4, issue 10, e7408 (1-10) (2009) 16

17 Introduction Usual way Whole mass spectrum (all pixels) Extract ion images Definition of Regions of Interest (RI) from individual ion images RIs Mass spectra Co(anti)-localization of lipids 17

18 Introduction Usual way Whole mass spectrum (all pixels) Extract ion images Definition of Regions of Interest (RI) from individual ion images RIs Mass spectra Co(anti)-localization of lipids Manual process strongly dependant on the operator subjectivity/experience 18

19 Introduction Aim: differentiate by TF-SIMS imaging CF mouse models from WT mices Samples: colon crypts 19

20 Introduction WT C16:1 FA (Lieberkühn glands) CF A B I J C D K L C18:0 FA (Lamina propria) Vit E C18:2 FA (Border) E F M N PI or ST fragment (mucosa) Cholesteryle Sulfate (Border) G H P 3-color overlay 20

21 Introduction WT A CF B C16:1 FA Different distributions of lipids (Lieberkühn C D histological substructures glands) I J between the K L No «clear» differences between CF and WT C18:2 FA samples C18:0 FA (Lamina propria) Vit E (Border) E F Strong inter-individual variations M N PI or ST fragment (mucosa) Cholesteryle Sulfate (Border) G H P 3-color overlay 21

22 Introduction STEP 2 multivariate PCA selection of PCs Principal Componen t Number Percent Variance Captured by PCA Model Eigenvalu e of Cov(X) % Variance Captured by this PC % Variance Captured Total

23 Introduction STEP 3: representing pixels in the space of scores PC1-PC2 plane Epithelial border Lieberkühn Glands Submucosa Lamina propria 23

24 24 Introduction STEP 4: partitioning clustering of PCA 1-4 selected variables 2 clusters 3 clusters 4 clusters 5 clusters

25 25 Introduction STEP 4: partitioning clustering of PCA 1-4 selected variables 2 clusters RESULTS 3 clusters - Well-defined regions of interest (RIs) - Selection of RIs for 7 WT and 6 CF samples allows their comparison by PCA - A Genetic Algorithm analysis of WT and CF samples provides a list of the most discriminating peaks 4 clusters 5 clusters

26 26 Introduction Ion mass (m/z) Ion Species Lipid family [CH 3 4 P].- PE, PC, SM fragment PI [C 4 H 11 N 4 P] - PC, SM fragment CS, ST fragment PI, ST C14:0 carboxylate Fatty Acid C16:0 carboxylate Fatty Acid [Vitamine E H]- VE [TG52:0 H]- TG

27 27 Introduction Lysosomal disease due to a lack of α-d-galactosidase-a (α-gala) of recessive transmission linked to the X chromosome (affects 1 over people) Accumulation of Gb 3 and Ga 2 glycosphingolipids in fluids and tissues H H H H H Structure of a Gb 3 (Trihexosylceramide) : H H H sidic sequence (a-gal/b-gal/b-glu) H H HN H Ga 2 (digalactosylceramide), Gal/Gal sequence Acyl sequence kidney deficiencies, neurological and cardiac evolutive lesions and angiokeratomas

28 28 Introduction Lysosomal disease due to a lack of α-d-galactosidase-a (α-gala) of recessive transmission linked to the X chromosome (affects 1 over people) Accumulation of Gb 3 and Ga 2 glycosphingolipids in fluids and tissues H H H H H Structure of a Gb 3 (Trihexosylceramide) : H H H sidic sequence (a-gal/b-gal/b-glu) H H HN H Ga 2 (digalactosylceramide), Gal/Gal sequence Acyl sequence kidney deficiencies, neurological and cardiac evolutive lesions and angiokeratomas

29 Introduction + Intensity (counts) No treatment of the sample 500x500 µm 2 256x256 pixels Bi 3+ primary ions Acquisition time 30 min Fluence ions.cm -2 Detection of very intense Ga 2 et Gb 3 signals Possible to localize a single species with a micrometer resolution Colocalization with vitamin E, cholesterol and cholesterol sulfate m/z D. Touboul et al., Int. J. Mass Spectrom. 2007, 260,

30 30 Introduction H N N H H N N H H N CH N H H N CH

31 31 Introduction Image size: 500x500 µm² Primary ions: Bi 3+, 25 kev Negative ion mode PIDD: ions.cm -2 Pixel size: 2x2 µm 2 Acquisition time: 30 min

32 32 Introduction

33 33 Introduction Artwork analysis Dogon art Rembrandt Grünewald

34 34 Introduction Temporal cortex

35 Introduction Variation of cholesterol distribution along the cortex (Alzheimer n=6, Controls n=4) A. N. Lazar et al. Acta Neuropathologica, 125, (2013) 35

36 Introduction Polybrominated flame retardants Endocrine disruptive chemicals Imaging of DBDE in organs of dosed rats vs. control ADRENALS ptical images D Dosed Control Cortex Medulla 2 mm E G F H 2 mm A. SeyerJ. Am. Soc. Mass Spectrom. 21, (2010), I 36

37 Introduction Polybrominated flame retardants Endocrine disruptive chemicals Imaging of DBDE in organs of dosed rats vs. control VARIES A Masson staining D Dosed G Control B 2 mm E H 2 mm -Accumulation in some of the numerous corpora lutea -Dependant on the corpus luteum s stage in the estrous cycle (?) 37

38 38 Introduction Used as preservative in eye drops of prostaglandin derivatives and betablockers as first line therapy for glaucoma But it is toxic: Damage of tissues due to oxidative stress Apoptosis

39 39 Introduction

40 40 Introduction CRNEA overlay N. Desbenoit et al. Anal. Bioanal. Chem., 405, (2013)

41 41 Introduction MALDI Imaging

42 42 Introduction TF-SIMS Imaging Drug Sphingomyelin Diglycerides Hair follicle colocalisation of the drug with the diglycerides around the hair follicles

43 43 Introduction Portrait of Nicolaes van Bambeeck Rembrandt van Rijn, 1641 Artwork analysis Rembrandt

44 44 Introduction Artwork analysis Rembrandt m/z 85 Canvas 1 st ground layer 2 nd ground layer Pictural layers Red grain 1500 μm long

45 45 Introduction m/z 85 (resin) 1500 μm long Amino acids m/z 59 (saccharide fragment: starch) Pb + Pb 2+ Pb 3+ Artwork analysis Rembrandt Lipid ions Lead white

46 46 Introduction Artwork analysis Rembrandt STRATIGRAPHY F THE SAMPLE Canvas Resin Umber Lead white Starch Lipid + lead Lake pigment

47 47 Introduction Artwork analysis Rembrandt

48 Introduction Artwork analysis Rembrandt Grunewald For tissue imaging, the micrometer lateral resolution is enough in many cases (SIMS) The mass resolution and mass accuracy should be as high as possible Many organic ions can be identified by their co-localization with specific fragments (SIMS) or by MS/MS (MALDI). Don t forget the gas phase dissociation chemistry! Identification by spectral comparisons with standard compounds is always useful The targeted compounds must be highly concentrated in welldefined areas to be imaged. In biology, the expertise of histologists and/or collaboration with clinicians is mandatory. In SIMS In situ identification by MS/MS is lacking complementarity with MALDI-MSMS. 48

49 49 Grants: Agence Nationale de la Recherche Scientifique (ANR) European Union (FP6 STREP Program) Institut de Chimie des Substances Naturelles (ICSN) Centre National de la Recherche Scientifique (CNRS)

50 50 Alain Brunelle David Touboul Vincent Guérineau Vanessa Petit Post-doc Mario llero Post-doc Nicolas Desbenoit Post-doc Alexandre Seyer PhD Farida Messouaf PhD