Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Similar presentations


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 Natalia Ivashikina – Use of Titanium Oxide - Metabolites Ulrike Koch/Lorrie Murphy/Anna Croft - Blood and Urine Metabolites Liz Allen - Single Cell Arabidopsis metabolites and correlating with NMR data Coffee 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 Barry Grail - (Michael Doenhoff) Peptides FT-ICR-MS Natalia Ivashikina – Metabolites Kareem Al Zubaidi – Single cell Tradescantia, Metabolites Mark Hooks – Nerve cell metabolites Paula Roberts – Metabolites 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 = Da Video

13 Neutron: ( ) ? Sucrose (-H): For M r 1000; 0.1 ppm = 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, for FT-MS

16 CH4methaneH NH3ammoniaH NH4ammoniumH CH2OformaldehydeH O2oxygenH H2Shydrogen sulfideH H2O2hydrogen peroxideH CH4methaneNa NH3ammoniaNa NH4ammoniumNa CO2 H NO2nitriteH CH2O2formateH CH2O2formic acidH C2H6OethanolH CH4SmethanethiolH CH2OformaldehydeNa O2oxygenNa CH4methaneK NH3ammoniaK H2Shydrogen sulfideNa H2O2hydrogen peroxideNa NH4ammoniumK C2H4O2acetaldehydeH C2H4O2acetic acidH C2H4O2glycolaldehydeH+

17 C5H4N4O2xanthineH C6H10O32-oxoisocaproic acidNa C5H12O5xylitolH C6H14N2ON-acetylputrescineNa C5H9NO2prolineK C5H9NO35-aminolevulinateNa C5H9NO3glutamate-5-semialdehydeNa C5H9NO3trans-4-hydroxy-L-prolineNa C7H7NO33-hydroxyanthranilateH C4H9N3O2creatineNa C6H13NO2isoleucineNa C6H13NO2leucineNa C8H11NO2dopamine and octopamineH C4H4O4fumaric acidK C4H4O5oxalacetic acidNa C5H8O3(R)-2-oxoisovalerateK C5H8O32-oxovaleric acidK C5H8O33-methyl-2-oxobutanoateK C5H8O4glutarateNa C5H8O4(R)-3-hydroxy-3-methyl-2-oxobutanoateMNa C5H8O42-acetolactateNa C5H8O4glutaric acidNa C7H6O4dihydroxybenzoic acidH C7H6O4gentisic acidH C4H8N2O3asparagineNa C6H12O2caproic acidK C5H12N2O2ornithineNa C4H7NO3aspartate-4-semialdehydeK C4H7NO4aspartic acidNa C5H11NO2valineK C6H9N3O2histidineH C4H6O4succinic acidK C4H6O5malic acidNa

18 C30H42N7O18P3Scoumaroyl-CoANa C31H44N7O19P3Sferuloyl-CoAH C28H46N7O19P3S6-carboxyhexanoyl-CoAK C55H92O7P2undecaprenyl diphosphateNa C27H44N7O20P3S(S)-3-hydroxy-3-methylglutaryl-CoAK C30H42N7O18P3Scoumaroyl-CoAK C30H42N7O19P3Scaffeoyl-CoANa C55H92O7P2undecaprenyl diphosphateK C31H44N7O19P3Sferuloyl-CoANa C30H42N7O19P3Scaffeoyl-CoAK C35H62N7O17P3Stetradecanoyl-CoAH C31H44N7O19P3Sferuloyl-CoAK C35H62N7O17P3Stetradecanoyl-CoANa C37H66N7O17P3Spalmitoyl-CoAH C35H62N7O17P3Stetradecanoyl-CoAK C37H66N7O17P3Spalmitoyl-CoANa C50H83NO21tomatineH C37H66N7O17P3Spalmitoyl-CoAK C50H83NO21tomatineNa C50H83NO21tomatineK

19 The Modern Trinity Genomics Proteomics Metabolomics

20 9.15 General Introduction. Deri Tomos 9.30 Technical Introduction. Barry Grail MALDI-TOF 9.45 Juma’a Al-Dulayymi – Mycolic acids Natalia Ivashikina – Use of Titanium Oxide - Metabolites Ulrike Koch/Lorrie Murphy/Anna Croft - Blood and Urine Metabolites Liz Allen - Single Cell Arabidopsis metabolites and correlating with NMR data Coffee 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 Barry Grail - (Michael Doenhoff) Peptides FT-ICR-MS Natalia Ivashikina – Metabolites Kareem Al Zubaidi – Single cell Tradescantia, Metabolites Mark Hooks – Nerve cell metabolites Paula Roberts – Metabolites 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: 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


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

Similar presentations


Ads by Google