Cryodetectors in Life Science Mass Spectrometry. Damian Twerenbold
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1 Cryodetectors in Life Science Mass Spectrometry Damian Twerenbold Institut de Physique de l'université Neuchâtel, Paul Scherrer Institut,Villigen GenSpec SA, Boudry Switzerland LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 1
2 Contents Genomics, Proteomics Mass Spectrometry Cryodetectors in Mass Spectrometry Status and Applications Outlook LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 2
3 Genomics and Proteomics Genomics: - DNA base sequence in Genome Database...-K-E-Q-V DNA bases 1 amino acid Proteomics: - Protein amino acid sequence in Protein Database LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 3
4 Proteomics: Reality is more complicated 2D-gel iso-electrical point apparent mass in complex biological systems: 1 gene 10 proteins post-translational modifications: (glycosylation, phosphorylation,... ) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 4
5 Goal of Genomics and Proteomics What do biologists want? ("biologist" = molecular biologists, biochemists, protein chemists, real biologists, pharmaceutical drug discoverers ) identify proteins: (partially) sequence primary structure and look up genomic and protein databases characterize proteins: analyze biologically relevant modifications look for protein complexes and networks: goal: biological function (new drug!) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 5
6 Analytical Instrumentation in Life Sciences ingredients: proteins, DNA, sugars, salts, fluids,... very complex mixtures Tasks: - preparation - separation - detection Analytical Methods: - chromatography - gel electrophoresis - mass spectrometry Biology -structure -pathways Bioinformatics: - Genomics and Proteomics Databases LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 6
7 Genomics, Proteomics Mass Spectrometry Cryodetectors in Mass Spectrometry Status and Applications Outlook LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 7
8 Time-of-Flight Mass Spectrometry TOF principle: separation according to velocity of molecules accelerating molecules to energy: E U = q U U kinetic energy: E kin =1/2 M v 2 E U M,q E kin E U = E kin tof = d M 2qU d detector NOTE: energy of molecule: E U = q U (no mass term! ) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 8
9 MALDI Molecule Launch MALDI: Matrix Assisted Laser Depletion & Ionization biomolecules embeded in laser light sensitive matrix laser energy absorbed by matrix mechanical momentum transfer of matrix molecules to the massive biomolecules laser pulse: laser pulse: 3 nsec, 10 5 Watt/cm 2 LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 9
10 Standard Ionizing Detectors Ionizing Detectors: detection mechanism: emission of secondary electrons from surface e - small quantum efficiency for slow molecules large and fast signal LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 10
11 Ionizing Detector Problem: Efficiency decrease of intensity: molecule launch? ionization? ion transmission? detector sensitivity? LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 11
12 Genomics, Proteomics Mass Spectrometry Cryodetectors in Mass Spectrometry Status and Applications Outlook LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 12
13 Molecule Detection with Cryodetectors Cryogenic Particle Detectors: detection mechanism: internal energy excitations (mev) protein (IgG) superconducting Niobium film Aluminium-oxid ( ( ( ( ( ( () ) ) ) ) ) epitaxial superconducting Tantal film sapphire substrate molecule impact energy deposition excess quasiparticles LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 13
14 Fundamentals of Calorimetric Detection density of states electronic and phonon excitations can be coupled to an electrical signal quasiparticles phonons electron-hole pairs energy super 1 mev E Debye 10 mev semi 1000 mev LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 14
15 History of Cryodetectors in Mass Spectrometry Neuchâtel 1996 Sn-junctions proof: basic principle Lawrence Livermore/ Lawrence Berkeley 1996 Nb-junctions proof: detector sensitivity 1000 times larger NIST Boulder / Neuchâtel 1997 microcalorimeter proof: charge deconvolution LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 15
16 Neuchâtel (1996) 50 µm x 50 µm Sn-STJ detector operated at 300 mk First Results: STJ mass IgG = 150'000 Da (antibody) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 16
17 Microcalorimeter collaboration (1998) NIST (Boulder) Institut Physique, Neuchâtel GenSpec SA 200 µm x 200 µm microcalorimeter operated at 100 mk molecule Ag-film hot electrons tunnel current Al-oxide Al-film G.Hilton,J.M.Martinis,D.A.Wollmann,K.D.Irwin,L.L.Dulcie, D.Gerber,P.M.Gillevet & D.Twerenbold, Nature, Vol 391, page 672, 12.February 1998 LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 17
18 Cryodetector versus Ionizing Detector equimolar PEG sample: IgG sample: Ionizing Detector Cryodetector (Al-junctions) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 18
19 Cryodetector versus Ionizing Detector LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 19
20 Energy as additional Information DNA 40-mer oligonucleotide time-of of-flight flight spectrum fragmentation all pulses only pulses with /-2 kev (same data set) kev pulseheight LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 20
21 Polystyrene 2000: laser power reduced laser power full laser power only neutrals (deflection plate) LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 21
22 Polystyrene 2000 LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 22
23 mass = 100 Da Mass Resolution detector time resolution: t = 10 nsec t = 100 nsec initial velocity dispersion: v 0 = 100 m/sec mass = 1 kda LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 23
24 Summary: Cryodetectors for MS pro: mass independent detection sensitivity high sensitivity for very large molecules pulse height proportional to molecule energy contra: clean up mass spectrum slow signal (100 nsec) low mass resolution at small mass molecules total overall sensitivity is still small small size of detectors LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 24
25 Genomics, Proteomics Mass Spectrometry Cryodetectors in Mass Spectrometry Status and Applications Outlook LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 25
26 Requirements for Cryodetectors no high energy resolution required! robust with respect to temperature fluctuations "molecules cannot go through thin windows" large area cryodetector (array technology) stable operation LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 26
27 PSI epi Ta STJ Detectors for Molecules fabricated at Paul Scherrer Institut 30 µm x 30 µm 180 µm epi Ta LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 27
28 Yale Voltage Biased Current Preamplifier 6 kev x-rays T base =0.32 K T STJ 0.6 K LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 28
29 Requirements for an Instrument Instrument should be "biologist" friendly (= fool proof) cryogenfree cryostat stable operation of cryodetectors (simple electronics) everything software controlled LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 29
30 Results with first Commercial Instrument - Comet Analytics, Flamatt, Switzerland - Product: Macromizer conventional MS-TOF Macromizer LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 30
31 Genomics, Proteomics Mass Spectrometry Cryodetectors in Mass Spectrometry Status and Applications Outlook LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 31
32 Life Science Community Reaction pro: fascinated to see single molecules mass independent detection high sensitivity for very large molecules contra: low mass resolution at small mass molecules total sensitivity (small detectors) promising applications protein complexes glycobiology biomarker discovery LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 32
33 Cryodetector Improvements The Life Science User's wish list: large area detector 1 cm 2 fast detector response 1 nsec "high" operating temperature 5 Kelvin LTD-10, Genova, 2003 Université Neuchâtel, Switzerland 33
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