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Bangor SBS Brucker Mass Spectrometers

Bangor SBS Brucker Mass Spectrometers. Brucker Reflex IV MALDI-TOF Brucker Daltonics Apex III FT-ICR-MS. Funded (~ 2001) by BBSRC Strategic Research Initiative Fund (SRIF) U Wales Reconfiguration Fund. For non-profit making or VAT- exempt research. Capital costs of instruments:

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Bangor SBS Brucker Mass Spectrometers

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  1. Bangor SBS Brucker Mass Spectrometers Brucker Reflex IV MALDI-TOF Brucker Daltonics Apex III FT-ICR-MS Funded (~ 2001) by BBSRC Strategic Research Initiative Fund (SRIF) U Wales Reconfiguration Fund For non-profit making or VAT- exempt research

  2. Capital costs of instruments: MALDI – £140,000MALDI Cooler – £10,000 FT - £473,000 Installation costs - considerable Running costs of instruments (without depreciation or technician) MALDI - £12,000 FT - £75,000 Current funding BBSRC (P uptake by rice) BBSRC (RELU) NERC (DU behaviour in soil) Wildlife DNA Ltd

  3. 9.15 General Introduction. Deri Tomos 9.30 Technical Introduction. Barry Grail MALDI-TOF 9.45  Juma’a Al-Dulayymi – Mycolic acids 10.10 Natalia Ivashikina – Use of Titanium Oxide - Metabolites 10.25 Ulrike Koch/Lorrie Murphy/Anna Croft - Blood and Urine Metabolites 10.50 Liz Allen - Single Cell Arabidopsis metabolites and correlating with NMR data 11.05 Coffee 11.25 Deri Tomos - i. (Honours Projects) Tea and Single cell salt-stressed Barley                     iii (Naoki Moritsuka) Soil solutes                     ii. (Sebastian Jäger) Chara corallina peptides and oligosaccharides 11.45 Barry Grail - (Michael Doenhoff) Peptides FT-ICR-MS12.00 Natalia Ivashikina – Metabolites 12.20 Kareem Al Zubaidi – Single cell Tradescantia, Metabolites 12.30 Mark Hooks – Nerve cell metabolites 12.40 Paula Roberts – Metabolites 12.50 Mike Hale – Wood degradation products 1.00  Anna Croft – Metabolites (IGER) 1.10  Sue Brittain – Soil analysis What next ?

  4. Mass spectroscopy One of the truly interdisciplinary methods in science. Extremely high sensitivity Can be applied to all physical states (solid, liquid, gas, plasma) High and low molecular mass. Often linked to another separating system (eg liquid chromatography) Determine Mass to Charge ratio (m/z) Ionise Separate in electric field Detect • Need to ionise • Electron ionisation • Chemical ionisation • Desorption ionisation (eg MALDI) • Spray ionisation (eg FT-ICR-MS) • Can run in positive or negative mode

  5. Matrix-assisted laser desorption ionisation – Time of Flight (MALDI TOF) Brucker Reflex IV MALDI-TOF

  6. Detectors Sample Laser (desorption) Ionisation Reflectron Time of Flight

  7. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) ions released into instrument Pulsed Nitrogen laser 337 nm energy transfer Matrix (absorption spectrum matches laser) Analyte

  8. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) ions released into instrument Pulsed Nitrogen laser 337 nm Titanium Oxide Matrix energy transfer Inorganic Matrix (Kinumi et al 2000) Negligible background Analyte

  9. Summary: Ionisation by photon (laser) desorption Very small volumes (pl) Separation by time of flight Detection Faraday cup ? Sensitive but relatively poor mass resolution

  10. Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS or FT-MS) Brucker Daltonics Apex III FT-ICR-MS

  11. Spray ionisation Hexapole Filter Cyclotron (cf Quadropole Filter) Direct injection or from LC system Non destructive detector Access for laser for in-detector fragmentation Dionex nano-lc system Central Control System Including Data acquisition and FT analysis

  12. Cyclotron resonance • Range of RF (ramped) • Induces new trajectory in each mass • Ions in new trajectory induce potential in detector plates Video Mass accuracy 0.1 ppm - for Mr ~ 1000 = 1234.1234 Da

  13. For Mr 1000; 0.1 ppm = 0.0001 dalton Neutron: 1.003717 (1.00678) ? Sucrose (-H): 341.10942 ? Mr = 329 Artefacts of Fourier Transformation ? Real peaks ?

  14. Summary: Ionisation by spray (heat or electric field) Very small volumes – nl/min Separation by hexapole and cyclotron Detection by image charge detection Sensitive and the best mass resolution

  15. Peak allocation (Prof Mike Burrell and colleagues, Sheffield University) Visual Basic (Excel) Some 500 plant and animal metabolites – entered by hand to 6 significant figures Negative mode assume (M - H+) Positive mode assume (M+ H+, K+or Na+) Choose resolution – eg 0.5 Da for MALDI, 0.005 for FT-MS

  16. The Modern Trinity Genomics Proteomics Metabolomics www.ne.jp/asahi/jun/icons/planche/trinite.html

  17. 9.15 General Introduction. Deri Tomos 9.30 Technical Introduction. Barry Grail MALDI-TOF 9.45  Juma’a Al-Dulayymi – Mycolic acids 10.10 Natalia Ivashikina – Use of Titanium Oxide - Metabolites 10.25 Ulrike Koch/Lorrie Murphy/Anna Croft - Blood and Urine Metabolites 10.50 Liz Allen - Single Cell Arabidopsis metabolites and correlating with NMR data 11.05 Coffee 11.25 Deri Tomos - i. (Honours Projects) Tea and Single cell salt-stressed Barley                     iii (Naoki Moritsuka) Soil solutes                     ii. (Sebastian Jäger) Chara corallina peptides and oligosaccharides 11.45 Barry Grail - (Michael Doenhoff) Peptides FT-ICR-MS12.00 Natalia Ivashikina – Metabolites 12.20 Kareem Al Zubaidi – Single cell Tradescantia, Metabolites 12.30 Mark Hooks – Nerve cell metabolites 12.40 Paula Roberts – Metabolites 12.50 Mike Hale – Wood degradation products 1.00  Anna Croft – Metabolites (IGER) 1.10  Sue Brittain – Soil analysis What next ?

  18. Deri Tomos - i. (Honours Projects) Tea and Single cell salt-stressed Barley                     iii (Naoki Moritsuka) Soil solutes                     ii. (Sebastian Jäger) Chara corallina peptides and oligosaccharides

  19. Ms Dunya Hurley. Tea infusions. (Honours Project) Use CsCl as normalising internal standard mixed with extract EGCG is (-) epigallocatechin gallate

  20. An obsession with micro capillaries ?

  21. An obsession with micro capillaries ?

  22. Ms Hannah Kemp. Single barley cells – salt stressed. (Honours Project) Use CsCl as normalising internal standard mixed with matrix

  23. Dr Naoki Moritsuka. Soil solutions (Proof of principle)

  24. Capillary zone electrophoresis Macroscopic soil solution samples NO3- Comparing analytical results of soil solution sampled from the root zone MALDI-TOF-MS (negative mode) MALDI-TOF-MS (positive mode) NO3- (K and Na salts) NO3-

  25. Mr Sebastian Jaeger and Dr Stephan Brandt . Single Cell Peptides Strands of Chara corallina grown in artifical pond water. Each strand consists of large cells connected at nodes. The marked cell is approximately 7 cm long (from Johnson, 2002)

  26. CHCA/IP/FA DD CHCA/TFA/ACN DD CHCA/TFA/ACN/NC DD CHCA/TFA/ACN/OGP DD Insulin ACTH Ribo nuc leaseA Myo glo bin Ubiquitin * Cyto chrome C * * • Figure : Comparison of MALDI-TOF spectra of pepmix8 samples obtained from different CHCA containing matrix solutions applying the dried droplet method. The matrix solutions consisted of isopropanole/formic acid (IP/FA), trifluoracetic acid/acetonitrile (TFA/ACN), trifluoracetic acid/acetonitrile/cellulose-nitrate (TFA/ACN/NC) and trifluoracetic acid/acetonitrile/octyl-glucopyranoside.(TFA/ACN/OGP). In the latter spectra the proteins of the pepmix8 are assigned to the corresponding peaks. Peaks marked with a star were produced by double charged ions. • . CHCA – hydroxy cyanocinnamic acid

  27. CHCA/TFA/ACN DD • CHCA/TFA/ACN/OGP DD CHCA/TFA/ACN/NC DD Figure : Comparison of MALDI-TOF spectra of single cell samples of Chara corallina obtained from different CHCA containing matrix solutions applying the dried droplet method. The matrix solutions consisted of trifluoracetic acid/acetonitrile (TFA/ACN), trifluoracetic acid/acetonitrile/octyl-glucopyranoside.(TFA/ACN/OGP) and trifluoracetic acid/acetonitrile/cellulose-nitrate (TFA/ACN/NC)

  28. SA/TFA/ACN SC SA/TFA/ACN SC SA/TFA/ACN/OGP SC SA/TFA/ACN/NC SC SA/TFA/ACN/NC SC Comparison of MALDI-TOF spectra of single cell samples of Chara corallina obtained from different sinapinic acid containing matrix solutions applying the slow crystallisation method. The matrix solutions consisted of trifluoracetic acid/acetonitrile (TFA/ACN), trifluoracetic acid/acetonitrile/octyl-glucopyranoside (TFA/ACN/OGP)and trifluoracetic acid/acetonitrile/cellulose-nitrate (TFA/ACN/NC).

  29. Enumeration of the repeated determined peaks and their approximated m/z-values in Dalton (Da) found in MALDI-TOF spectra of Chara corallina single cell samples applying sinapinic acid containing matrices. Generally detected masses are written in italics, whereas frequently found one are written in normal types. (Jäger (2003)) Subsequently Stephan Brandt repeated this approach with samples treated with: Proteinase K Amylase Protoplasting mixture (Cellulase and Macerozyme) RNAse/DNAse Range of characteristics – some totally resistant - some digested by proteinase and amylase

  30. Dr Karim Al-Zubaidi. Single leaf cells – Tradescantia virginiana (Proof of concept) Nanospray allows direct injection of single cell samples 10 mm Cell volumes samples > 10 – 100 pl NanospraysSamples: > 100 – 200 nl

  31. An obsession with micro capillaries ?

  32. SiCSA Single Cell Sampling and Analysis Picolitre osmometry Fluorescent microscopy Immunoassay Capillary electrophoresis X-ray microanalysis

  33. Tradescantia SiCSA -ve ion. Nanospray 50% Acetonitrile, 0.1% Triethylamine, 1% Chloroform Sucrose ? /Data/bandat05/jun_05/20_jun_05/scsneg/2/pdata/1 Administrator Tue Oct 18 09:28:14 2005 Sample from single Tradescantia leaf mesophyll cell (SiCSA)

  34. Natalia Ivashikina Tradescantia epidermal cells Glucose Glucose Aconitate ? Malate Aconitate ? Sucrose Malate Arg Citrate Arginine Glucose Glucose Aconitate ? Malate Aconitate ? Malate Citrate Sucrose Sucrose Arginine Arginine Citrate

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