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Bangor SBS Brucker Mass Spectrometers Brucker Reflex IV MALDI-TOF Brucker Daltonics Apex III FT-ICR-MS Funded (~ 2001) by BBSRC Strategic Research Initiative.

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Presentation on theme: "Bangor SBS Brucker Mass Spectrometers Brucker Reflex IV MALDI-TOF Brucker Daltonics Apex III FT-ICR-MS Funded (~ 2001) by BBSRC Strategic Research Initiative."— Presentation transcript:

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 – £140,000 MALDI 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-MS 12.00 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) Need to ionise Electron ionisation Chemical ionisation Desorption ionisation (eg MALDI) Spray ionisation (eg FT-ICR-MS) Can run in positive or negative mode Ionise Separate in electric field Detect

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

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

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

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

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

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

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

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

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 17.03857616.0313CH4methaneH+ 18.03382517.026549NH3ammoniaH+ 19.0416518.034374NH4ammoniumH+ 31.017840630.0105646CH2OformaldehydeH+ 32.997105231.9898292O2oxygenH+ 34.99499733.987721H2Shydrogen sulfideH+ 35.012755234.0054792H2O2hydrogen peroxideH+ 39.020521416.0313CH4methaneNa 40.015770417.026549NH3ammoniaNa 41.023595418.034374NH4ammoniumNa 44.997105243.9898292CO2 H+ 47.000179245.9929032NO2nitriteH+ 47.012755246.0054792CH2O2formateH+ 47.012755246.0054792CH2O2formic acidH+ 47.049140646.0418646C2H6OethanolH+ 49.01064748.003371CH4SmethanethiolH+ 52.99978630.0105646CH2OformaldehydeNa 54.979050631.9898292O2oxygenNa 54.994458416.0313CH4methaneK 55.989707417.026549NH3ammoniaK 56.976942433.987721H2Shydrogen sulfideNa 56.994700634.0054792H2O2hydrogen peroxideNa 56.997532418.034374NH4ammoniumK 61.028405260.0211292C2H4O2acetaldehydeH+ 61.028405260.0211292C2H4O2acetic acidH+ 61.028405260.0211292C2H4O2glycolaldehydeH+

17 153.0407012152.0334252C5H4N4O2xanthineH+ 153.0522152130.0629938C6H10O32-oxoisocaproic acidNa 153.075749152.068473C5H12O5xylitolH+ 153.099834130.1106126C6H14N2ON-acetylputrescineNa 154.0264866115.0633282C5H9NO2prolineK 154.0474642131.0582428C5H9NO35-aminolevulinateNa 154.0474642131.0582428C5H9NO3glutamate-5-semialdehydeNa 154.0474642131.0582428C5H9NO3trans-4-hydroxy-L-prolineNa 154.0498688153.0425928C7H7NO33-hydroxyanthranilateH+ 154.0586976131.0694762C4H9N3O2creatineNa 154.0838496131.0946282C6H13NO2isoleucineNa 154.0838496131.0946282C6H13NO2leucineNa 154.0862542153.0789782C8H11NO2dopamine and octopamineH+ 154.9741168116.0109584C4H4O4fumaric acidK 154.9950944132.005873C4H4O5oxalacetic acidNa 155.0105022116.0473438C5H8O3(R)-2-oxoisovalerateK 155.0105022116.0473438C5H8O32-oxovaleric acidK 155.0105022116.0473438C5H8O33-methyl-2-oxobutanoateK 155.0314798132.0422584C5H8O4glutarateNa 155.0314798132.0422584C5H8O4(R)-3-hydroxy-3-methyl-2-oxobutanoateMNa 155.0314798132.0422584C5H8O42-acetolactateNa 155.0314798132.0422584C5H8O4glutaric acidNa 155.0338844154.0266084C7H6O4dihydroxybenzoic acidH+ 155.0338844154.0266084C7H6O4gentisic acidH+ 155.0427132132.0534918C4H8N2O3asparagineNa 155.0468876116.0837292C6H12O2caproic acidK 155.0790986132.0898772C5H12N2O2ornithineNa 156.0057512117.0425928C4H7NO3aspartate-4-semialdehydeK 156.0267288133.0375074C4H7NO4aspartic acidNa 156.0421366117.0789782C5H11NO2valineK 156.0767522155.0694762C6H9N3O2histidineH+ 156.9897668118.0266084C4H6O4succinic acidK 157.0107444134.021523C4H6O5malic acidNa

18 936.141206913.1519848C30H42N7O18P3Scoumaroyl-CoANa 944.169825943.1625494C31H44N7O19P3Sferuloyl-CoAH+ 948.141358909.1781994C28H46N7O19P3S6-carboxyhexanoyl-CoAK 949.621046926.6318242C55H92O7P2undecaprenyl diphosphateNa 950.120622911.157464C27H44N7O20P3S(S)-3-hydroxy-3-methylglutaryl-CoAK 952.115143913.1519848C30H42N7O18P3Scoumaroyl-CoAK 952.136121929.1468994C30H42N7O19P3Scaffeoyl-CoANa 965.594983926.6318242C55H92O7P2undecaprenyl diphosphateK 966.151771943.1625494C31H44N7O19P3Sferuloyl-CoANa 968.110058929.1468994C30H42N7O19P3Scaffeoyl-CoAK 978.320846977.3135702C35H62N7O17P3Stetradecanoyl-CoAH+ 982.125708943.1625494C31H44N7O19P3Sferuloyl-CoAK 1000.30279977.3135702C35H62N7O17P3Stetradecanoyl-CoANa 1006.352151005.34487C37H66N7O17P3Spalmitoyl-CoAH+ 1016.27673977.3135702C35H62N7O17P3Stetradecanoyl-CoAK 1028.334091005.34487C37H66N7O17P3Spalmitoyl-CoANa 1034.553031033.545756C50H83NO21tomatineH+ 1044.308031005.34487C37H66N7O17P3Spalmitoyl-CoAK 1056.534981033.545756C50H83NO21tomatineNa 1072.508911033.545756C50H83NO21tomatineK

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

20 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-MS 12.00 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 ?

21

22 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

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

24 An obsession with micro capillaries ?

25

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

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

28 NO 3 - MALDI-TOF-MS (negative mode)MALDI-TOF-MS (positive mode) Comparing analytical results of soil solution sampled from the root zone Capillary zone electrophoresis NO 3 - (K and Na salts) Macroscopic soil solution samples

29 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)

30 CHCA/IP/FA DD CHCA/TFA/ACN DD CHCA/TFA/ACN/NC DD CHCA/TFA/ACN/OGP DD ACTH ACTH Insulin U bi qu iti n * Cyto chrom e C Ribo nuc lease A * Myo glo bin * 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

31 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/AC N/OGP) and trifluoracetic acid/acetonitrile/cellulose- nitrate (TFA/ACN/NC)

32 SA/TFA/ACN SC SA/TFA/ACN/O GP 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).

33 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

34

35 10 mm Nanospray allows direct injection of single cell samples Cell volumes samples > 10 – 100 pl NanospraysSamples: > 100 – 200 nl Dr Karim Al-Zubaidi. Single leaf cells – Tradescantia virginiana (Proof of concept)

36 An obsession with micro capillaries ?

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

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

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


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