Bio and Polymer Analytics. RD Instrumental Analytical Chemistry. Organic Trace Analytics. RD Environmental & Process Analytics

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1 MOLEKULARE IMAGING MASSENSPEKTROMETRIE VON GEWEBSOBERFLÄCHEN und BIOPOLYMERANALTIK Günter Allmaier RESEARCH GROUP BIO- AND POLYMER ANALYSIS Vienna University of Technology, Institute of Chemical Technologies and Analytics, Vienna, Austria ORGANISATION UNITS OF IN BIOANALYTICS INVOLVED RD Instrumental Analytical Chemistry Univ. Prof. Dr. G. Allmaier RD Environmental & Process Analytics AO. Univ. Prof. Dr. B. Lendl Process Analytics AO. Univ. Prof. Dr. B. Lendl Bio and Polymer Analytics Univ. Prof. Dr. G. Allmaier Organic Trace Analytics AO. Univ. Prof. Dr. E. Rosenberg Micro and Nano Analytics AO. Univ. Prof. Dr. G. Friedbacher Physical Analytics AO. Univ. Prof. Dr. H. Hutter Lab of Metabolomics & Bioprocess Analytics Ass. Prof. Dr. M. Marchetti-Deschmann Atomic Spectroscopy Priv. Doz. Dr. A. Limbeck 1

2 WHAT IS LINEAR TOF MASS SPECTROMETRY? A HIGH VACUUM ION SEPARATION TECHNIQUE WITH THEORETICAL UNLIMITED m/z RANGE Start when the laser hits you! How long I have to move? Finished in nano seconds! vmaldi LIN AND RTOF MS MCP detector Flight tube turbo CF Reflector MCP detector LinTOF MS Source ion optics MALDI target SAC turbo 2

3 BIOIMAGING TECHNIQUES Single molecule imaging (MRI, X-ray, CT, FRET, TEM, AFM, ) Spectroscopy imaging (FT-IR, Raman, Scanning near-field optical microscopy, Fluorescence ) Mass spectrometry imaging (SIMS, ME- SIMS, MALDI, DESI, ) PROFILING MS Low Density Droplet Array Slice frozen tissue on cryostat (~12 μm thick) or thin object of interest Apply matrix Thaw slice onto MALDI plate, allow to dry IMAGING MS Homogeneous coating Acquire mass spectra Molecular profiles Molecular Images m/z

4 NATURAL LATEX AS MEDICAL DEVICES AND IN OTHER APPLICATIONS CONNECTED WITH ALLERGIES Typ I allergies MOLECULAR IMAGING MASS SPECTROMETRY TO DETERMINE THE LATERAL DISTRIBUTION OF PROTEIN ALLERGENS IN LATEX WITHOUT LABELING 4

5 DISTRIBUTION OF MINOR HEVEIN ALLERGEN ON MEDICAL LATEX GLOVES Outer Surface m/z m/z Inner Surface White, highest local concentration Dark red and black, lowest concentration or not present ANALYSIS OF DRUGS AND METABOLITES IN TISSUE BY MASS SPECTROMETRYRY Cut frozen slice (12 µm) Dose animal orally or i.v. Remove tissue Apply MALDI matrix Analyze by: MALDI MS and MS/MS 5

6 WHOLE MOUSE IMAGING MASS SPECTROMETRY TU Courtesy Stoeckli (Novartis) DISTRIBUTION OF SMALL MOLECULES AS LIPIDS IN SLICES OF HUMAN KNEE EXPLANTATE BY IMAGING MASS SPECTROMETRY femoralcomponent (cobalt-chromate) PE-UHMW surface synovial fluid stemmed tibial plate (cobalt-chromate) Molecule with m/z 256 6

7 2-DIMENSIONAL IMAGING MASS SPECTROMETRY AT ATMOSPHERIC PRESSURE - DESI SOURCE ATTACHED TO IM-QqRTOF January 2010 at Vienna University of Technology (1st device in Europe) CHEMICAL COMPOSITION OF THE CONSTITUENT OF RULIDE TABLET (ROXITHROMYCIN, MACROLIDE ANTIBIOTIC) OD 8 mm Height 3 mm 7

8 DISTRIBUTION OF ACTIVE DRUG (m/z ) ON THE SURFACE OF THE COMPLETE RULIDE TABLET BY DESI IMAGING MASS SPECTROMETRY 8 mm The red spot is the area, where tuning and optimization was performed. SAMPLE PREPARTION FROM CULTIVATION PLATE SMEAR TECHNIQUE WITH NANOCOATED SS POLYMER TARGET Select from blood agar plate and transfer a single colony (approx microrgansisms) disposable loop Deposit sample by means of the loop onto the disposable microscope format slide Add05µl 0.5 matrix solution and mix 8

9 Voyager Spec #1=>AdvBC(32,0.5,1.0)=>NF0.7[BP = , 2117] Mass (m/z) FUSION OF PHYSIOLOGY AND PROTEOMICS FOR MICROORGANISM ID Protein Bacterial cell Physiological parameters (e.g. metabolic activity, enzyme hydrolysis, growth in the presence of inhibitors, acidification) Identity % Inte ensity IC/ISMS VITEK-2 One isolate (sample) Two HTP techniques with db Identity 17 ATTACHMENT OF AN ULTRA HIGH-MASS (UHM) DETECTOR ON STANDARD MALDI TOF MASS SPECTROMETER 9

10 BASIC STRUCTURE OF IMMUNGLOBULIN M (Ig M) PENTAMERIC FORM (largest monodisperse glycoprotein, % CHO content, sometimes impurities of hexameric form) MALDI MASS SPECTRUM OF Ig M WITH UHM DETECTOR AND EXTENDED SOFTWARE 100 %Int. 3+ [M] [M] 2+ MALDI matrix: SA, 500 unselected laser pulses, smoothed Gauss 500 MWt. 981 kda ([M] n+ (n = 1-4)) [M] 5+ [M] + Up to 2 million Da or 2 MegaDa! Mass/Charge

11 WHAT IS GEMMA? Gemma (Lower Austrian dialect) Let s go! GEMMA (acronym) Gas-phase Electrophoretic Mobility Molecular Analyzer MODULAR GEMMA SYSTEM FOR NANO PARTICLE DIAMETER DETERMINATION 2.5 nm 80 nm (EXTENSION UP TO 300 nm) 2006 at Vienna University of Technology (1st device in Europe) ENPS Molecular weight determination from 3 kda to 10 MDa (10000 kda) 11

12 GEMMA OF HUMANE RHINOVIRUS (SINGLE-STRANDED STRANDED RNA VIRUS) TYPE 2 AVERAGE VIRUS SIZE nm EMD SIZE DETERMINATION OF CANDIDATE VACCINE BY GEMMA AND COLLECTION BY ENPS ~ 46 nm Sampling for DotBlot Particle concentration [Particlel/cm³] Sampling on mica for AFM Particle diameter [nm] 12

13 DAS TEAM 13