Bildgebende Massenspektrometrie Erfolgreiche Anwendungen in der biomedizinischen Forschung. Markus Stoeckli
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1 Bildgebende Massenspektrometrie Erfolgreiche Anwendungen in der biomedizinischen Forschung Markus Stoeckli
2 MALDI Mass Spectrometric Imaging Principle 2 MALDI MSI Markus Stoeckli September 2014
3 Comparison of Human Lung Tumor Blots Squamous Cell Carcinoma Squamous Cell Carcinoma Adenocarcinoma Mass (m/z) 3 MALDI MSI Markus Stoeckli September 2014
4 Peptides on Mouse Brain Tissue 4 MALDI MSI Markus Stoeckli September 2014
5 What s wrong with this slide? 5 MALDI MSI Markus Stoeckli September 2014
6 The Vision in MALDI MSI Markus Stoeckli September 2014
7 Drug Discovery Process Source: 7 MALDI MSI Markus Stoeckli September 2014
8 Amyloid Beta Peptides Aß-(1-40) Aß-(1-42) 8 MALDI MSI Markus Stoeckli September 2014 stained
9 Cyclic Peptide Intra Muscular Deposit, 1 week post dose 9 MALDI MSI Markus Stoeckli September 2014
10 Proteins and MS Imaging some difficulties Membrane Proteins Source: Ghaemmaghami, Nature (2003), 425, MALDI MSI Markus Stoeckli September 2014
11 More about Proteins Separation is key High concentration range Low MS sensitivity in high mass range Suppression effect during ionization Targeted Px Extraction, digest, fractionation, separation, SRM Source: Picotti, Nat. Methods, 2008, 5 (11), MALDI MSI Markus Stoeckli September 2014
12 MSI on Proteins A Reality Check Data from IMSC 2014 MS Imaging On-tissue digest, MALDI TOF 250 peptides, 109 proteins (Bruker, Arndt Asperger) LC/MS DIA, variable width 20 k peptides, 2000 proteins (Ying Zhang) HILIC, fractionations: 7000 proteins (Nathalie Selevsek) 12 MALDI MSI Markus Stoeckli September 2014
13 Small Molecules and MSI Background LCMS dissection of animal limited spatial resolution only parts of animal WBAL label required total radioactivity not specific 13 MALDI MSI Markus Stoeckli September 2014
14 MALDI MS Imaging Platform Current setup at NIBR Animal Dosing Sectioning Matrix Coating Data Acquisition Data Cube Generation Data Processing and Visualization 14 MALDI MSI Markus Stoeckli September 2014
15 Compound Distribution 24 h post dose, MRM 15 MALDI MSI Markus Stoeckli September 2014
16 Hydroxylated Compound Distribution 24 h post dose, MRM 16 MALDI MSI Markus Stoeckli September 2014
17 LLOD/LLOQ Signal R² = Tissue Concentration [um] 17 MALDI MSI Markus Stoeckli September 2014
18 MS Imaging Workflow Compound and metabolite distributions Request from Team MS Method Development LLOD on Liver Dosing Matrix Deposition Dehydration & Formatting Transfer Embedding & Sectioning Data Acquisition Data Processing Reporting 18 MALDI MSI Markus Stoeckli September 2014
19 Workflow and Timelines Typical times, depending on experimental design 1 week Identification of compound & external standard Request background info (PK data) and compound LLOD of compound on plate and tissue, MS/MS method (2 d) Write study protocol (1/2 d) Dosing (data, organization, treatment, sacrifice, freezing, sectioning, 5 to 8 d) Measurement of sections (5 to 10 d) Data evaluation (5 to 15 d) 19 MALDI MSI Markus Stoeckli September 2014
20 Automation 20 MALDI MSI Markus Stoeckli September 2014
21 Matrix Deposition Artifacts 21 MALDI MSI Markus Stoeckli September 2014
22 imatrixspray Free and open source software and hardware Fully automated Multiple reagents Optimized for MALDI MSI Automation friendly imatrixspray.com 22 MALDI MSI Markus Stoeckli September 2014
23 High Mass Resolution and Specificity OrbiTrap, resolution FlashQuant, SRM 23 MALDI MSI Markus Stoeckli September 2014
24 Time Series 8 h M1 (533 > 446) Cpd (535 > 448) M2 (551 > 449) M3 (551 > 464) M4 (727 > 551) Optical 24 MALDI MSI Markus Stoeckli September 2014
25 Time Series 24 h 25 MALDI MSI Markus Stoeckli September 2014
26 Time Series 48 h Optical 26 MALDI MSI Markus Stoeckli September 2014
27 Time Series 72 h Optical 27 MALDI MSI Markus Stoeckli September 2014
28 Quantification Methods Internal Standard 4. MALDI Matrix 3. Internal Standard (Isotope or similar) 2. External Standard or Spiked Tissue 1. Tissue Section of Dosed Animal c I I Cpd IntStd I I IntStd ExtStd c ExtStd 28 MALDI MSI Markus Stoeckli September 2014
29 Data Evaluation and Reporting 3h, 10 mg/kg, 3 h p.o., 3 separate sections from same animal c (i) [µm] MALDI MSI Markus Stoeckli September 2014
30 Direct Comparison QWBA/MSI BT-474 tumor-bearing nude mice 10 mg/kg [ 14 C]-labeled compound Brain [um] 15 Lung h 2h 4h 8h 0 0.5h 2h 4h 8h [um] 50 Liver [um] 40 Tumor h 2h 4h 8h 0 0.5h 2h 4h 8h 30 MALDI MSI Markus Stoeckli September 2014
31 WBAL Compared to MALDI 10 mg/kg p.o., 2 h post dose WBAL Kidney Tumor Lung Brain Compound Concentration Bile Liver compound / desmethyl MALDI MSI Markus Stoeckli September 2014
32 Alternatives to MALDI MSI Sensitivity is key Source: M.-Z. Huang et al. / Analytica Chimica Acta 702 (2011) MALDI MSI Markus Stoeckli September 2014
33 Surface Sampling Tissue concentrations by liquid extraction from tissue surface Compound Metabolite MSi LESA 0 blood brain liver stomach content 33 MALDI MSI Markus Stoeckli September 2014
34 WBAL Brain Penetration 50 um spatial resolution, nm sensitivity Source: Esther von Kerkhof, Novartis 34 MALDI MSI Markus Stoeckli September 2014
35 PET/CT of Biologics Quantification, Resolution - PK, Biodistribution 124 I-labeled Ab fragment 64 Cu-DOTA-labeled Ab frag. 35 MALDI MSI Markus Stoeckli September 2014
36 Lung Sections of TB-Infected Rabbits 30 µm Pixel Imaging of PA824 in Granuloma 36 MALDI MSI Markus Stoeckli September 2014 Brendan Prideaux
37 PE ** Compound Optical Scalp Biopsy Sections measure by SIMS High resolution Control PG/OL CSF Nanoparticles PG MALDI MSI Markus Stoeckli September 2014
38 A starting point for MS imaging 38 MALDI MSI Markus Stoeckli September 2014
39 Acknowledgment Prakash Mistry Fabian Von Arx Fabienne Schaeffer Robert Cozens Francesco Hofmann Masato Murakami Dieter Staab Brendan Prideaux Nicole Ehrhard Gregory Morandi Rocco Falchetto Steve Martin ABSciex 39 MALDI MSI Markus Stoeckli September 2014
40 40 MALDI MSI Markus Stoeckli September 2014
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