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:

MALDI – £140,000MALDI Cooler – £10,000

FT - £473,000

Installation costs - considerable

Running costs of instruments (without depreciation or technician)

MALDI - £12,000FT - £75,000

Current fundingBBSRC (P uptake by rice)BBSRC (RELU)NERC (DU behaviour in soil)Wildlife DNA Ltd

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 ?

Mass spectroscopy

One of the truly interdisciplinary methods in science.Extremely high sensitivityCan 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 ionisationChemical ionisationDesorption ionisation (eg MALDI)Spray ionisation (eg FT-ICR-MS)

Can run in positive or negative mode

Ionise Separate in electric field Detect

Matrix-assisted laser desorption ionisation – Time of Flight (MALDI TOF)

Brucker Reflex IV MALDI-TOF

Laser (desorption) Ionisation

Sample

Time of Flight

Reflectron

Detectors

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

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

Summary:Ionisation by photon (laser) desorptionVery small volumes (pl)Separation by time of flightDetection Faraday cup ?

Sensitive but relatively poor mass resolution

Brucker Daltonics Apex III FT-ICR-MS

Fourier Transform Ion Cyclotron Resonance Mass Spectrometer(FT-ICR-MS or FT-MS)

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 SystemIncluding Data acquisition and FT analysis

• 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 Mr ~ 1000 = 1234.1234 Da

Video

Neutron: 1.003717 (1.00678) ?Sucrose (-H): 341.10942

For Mr 1000; 0.1 ppm = 0.0001 dalton

?

Real peaks ?Artefacts of

Fourier Transformation ?

Mr = 329

Summary:Ionisation by spray (heat or electric field)Very small volumes – nl/minSeparation by hexapole and cyclotronDetection by image charge detection

Sensitive and the best mass resolution

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

17.038576 16.0313 CH4 methane H+

18.033825 17.026549 NH3 ammonia H+

19.04165 18.034374 NH4 ammonium H+

31.0178406 30.0105646 CH2O formaldehyde H+

32.9971052 31.9898292 O2 oxygen H+

34.994997 33.987721 H2S hydrogen sulfide H+

35.0127552 34.0054792 H2O2 hydrogen peroxide H+

39.0205214 16.0313 CH4 methane Na

40.0157704 17.026549 NH3 ammonia Na

41.0235954 18.034374 NH4 ammonium Na

44.9971052 43.9898292 CO2 CO2 H+

47.0001792 45.9929032 NO2 nitrite H+

47.0127552 46.0054792 CH2O2 formate H+

47.0127552 46.0054792 CH2O2 formic acid H+

47.0491406 46.0418646 C2H6O ethanol H+

49.010647 48.003371 CH4S methanethiol H+

52.999786 30.0105646 CH2O formaldehyde Na

54.9790506 31.9898292 O2 oxygen Na

54.9944584 16.0313 CH4 methane K

55.9897074 17.026549 NH3 ammonia K

56.9769424 33.987721 H2S hydrogen sulfide Na

56.9947006 34.0054792 H2O2 hydrogen peroxide Na

56.9975324 18.034374 NH4 ammonium K

61.0284052 60.0211292 C2H4O2 acetaldehyde H+

61.0284052 60.0211292 C2H4O2 acetic acid H+

61.0284052 60.0211292 C2H4O2 glycolaldehyde H+

153.0407012 152.0334252 C5H4N4O2 xanthine H+

153.0522152 130.0629938 C6H10O3 2-oxoisocaproic acid Na

153.075749 152.068473 C5H12O5 xylitol H+

153.099834 130.1106126 C6H14N2O N-acetylputrescine Na

154.0264866 115.0633282 C5H9NO2 proline K

154.0474642 131.0582428 C5H9NO3 5-aminolevulinate Na

154.0474642 131.0582428 C5H9NO3 glutamate-5-semialdehyde Na

154.0474642 131.0582428 C5H9NO3 trans-4-hydroxy-L-proline Na

154.0498688 153.0425928 C7H7NO3 3-hydroxyanthranilate H+

154.0586976 131.0694762 C4H9N3O2 creatine Na

154.0838496 131.0946282 C6H13NO2 isoleucine Na

154.0838496 131.0946282 C6H13NO2 leucine Na

154.0862542 153.0789782 C8H11NO2 dopamine and octopamine H+

154.9741168 116.0109584 C4H4O4 fumaric acid K

154.9950944 132.005873 C4H4O5 oxalacetic acid Na

155.0105022 116.0473438 C5H8O3 (R)-2-oxoisovalerate K

155.0105022 116.0473438 C5H8O3 2-oxovaleric acid K

155.0105022 116.0473438 C5H8O3 3-methyl-2-oxobutanoate K

155.0314798 132.0422584 C5H8O4 glutarate Na

155.0314798 132.0422584 C5H8O4 (R)-3-hydroxy-3-methyl-2-oxobutanoateM Na

155.0314798 132.0422584 C5H8O4 2-acetolactate Na

155.0314798 132.0422584 C5H8O4 glutaric acid Na

155.0338844 154.0266084 C7H6O4 dihydroxybenzoic acid H+

155.0338844 154.0266084 C7H6O4 gentisic acid H+

155.0427132 132.0534918 C4H8N2O3 asparagine Na

155.0468876 116.0837292 C6H12O2 caproic acid K

155.0790986 132.0898772 C5H12N2O2 ornithine Na

156.0057512 117.0425928 C4H7NO3 aspartate-4-semialdehyde K

156.0267288 133.0375074 C4H7NO4 aspartic acid Na

156.0421366 117.0789782 C5H11NO2 valine K

156.0767522 155.0694762 C6H9N3O2 histidine H+

156.9897668 118.0266084 C4H6O4 succinic acid K

157.0107444 134.021523 C4H6O5 malic acid Na

936.141206 913.1519848 C30H42N7O18P3S coumaroyl-CoA Na

944.169825 943.1625494 C31H44N7O19P3S feruloyl-CoA H+

948.141358 909.1781994 C28H46N7O19P3S 6-carboxyhexanoyl-CoA K

949.621046 926.6318242 C55H92O7P2 undecaprenyl diphosphate Na

950.120622 911.157464 C27H44N7O20P3S (S)-3-hydroxy-3-methylglutaryl-CoA K

952.115143 913.1519848 C30H42N7O18P3S coumaroyl-CoA K

952.136121 929.1468994 C30H42N7O19P3S caffeoyl-CoA Na

965.594983 926.6318242 C55H92O7P2 undecaprenyl diphosphate K

966.151771 943.1625494 C31H44N7O19P3S feruloyl-CoA Na

968.110058 929.1468994 C30H42N7O19P3S caffeoyl-CoA K

978.320846 977.3135702 C35H62N7O17P3S tetradecanoyl-CoA H+

982.125708 943.1625494 C31H44N7O19P3S feruloyl-CoA K

1000.30279 977.3135702 C35H62N7O17P3S tetradecanoyl-CoA Na

1006.35215 1005.34487 C37H66N7O17P3S palmitoyl-CoA H+

1016.27673 977.3135702 C35H62N7O17P3S tetradecanoyl-CoA K

1028.33409 1005.34487 C37H66N7O17P3S palmitoyl-CoA Na

1034.55303 1033.545756 C50H83NO21 tomatine H+

1044.30803 1005.34487 C37H66N7O17P3S palmitoyl-CoA K

1056.53498 1033.545756 C50H83NO21 tomatine Na

1072.50891 1033.545756 C50H83NO21 tomatine K

The Modern Trinity

GenomicsProteomics

ww

w.ne.jp/asahi/jun/icons/planche/trinite.htm

l

Metabolomics

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 ?

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

Ms Dunya Hurley. Tea infusions. (Honours Project)

Use CsCl as normalising internal standard mixed with extract

EGCG is (-) epigallocatechin gallate

An obsession with micro capillaries ?

An obsession with micro capillaries ?

Ms Hannah Kemp. Single barley cells – salt stressed. (Honours Project)

Use CsCl as normalising internal standard mixed with matrix

Dr Naoki Moritsuka. Soil solutions

(Proof of principle)

0 50 100 150 200 250 300 350 m /z

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a.i.

/D = /data_06/naoki-sam a-data/N P _1/0_A 5_1S R ef/pdata/1 A dm inistrator M on Jul 17 11:44:20 2006

0 50 100 150 200 250 300 350 m /z

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/D = /data_06/naoki-sam a-data/N P _1_positive/0_A 5_1S R ef/pdata/1 A dm inistrator M on Jul 17 11:45:25 2006

0

5

10

15

20

25

0 2 4 6 8 10Time (min)

Sig

nal

(m

V)

NO3-

NO3-

MALDI-TOF-MS (negative mode) MALDI-TOF-MS (positive mode)

Comparing analytical results of soil solution sampled from the root zone

Capillary zone electrophoresis

NO3- (K and Na salts)

Macroscopic soil solution samples

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)

50 00 10 000 15 000 m /z

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/D = /da ta/N e w /d eri_ ca ra /21 _11 _02 /pe pm ix8A 1 /0_A 1_1 S Lin/p data/1 A dm in istrato r F ri N ov 22 09 :39:39 200 2

CHCA/IP/FA DD

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 1 6 0 0 0 m /z

0

2 0 0 0

4 0 0 0

6 0 0 0

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1 0 0 0 0

a .i.

/D = /d a ta /N e w /d e ri_ ca ra /2 1 _ 1 1 _ 0 2 /p e p m ix8 A 7 /0 _ A 7 _ 1 S L in /p d a ta /1 A d m in istra to r F ri N o v 2 2 1 0 :1 4 :1 7 2 0 0 2

CHCA/TFA/ACN DD

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/D = /data/N ew /deri_cara/29_11_02/I3/0_I3_1S Lin/pdata/1 A dm inistrator F ri N ov 29 17:41:53 2002

CHCA/TFA/ACN/NC DD

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/D = /data/N ew /deri_cara/21_11_02/pepm ix8A 10/0_A 10_1S Lin/pdata/1 A dm inistrator F ri N ov 22 09:49:41 2002

CHCA/TFA/ACN/OGP DD

40 00 6 0 0 0 8 0 0 0 1 0 0 0 0 12 000 14 000 16 000 m /z

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/D = /da ta/N e w /d eri_ ca ra /29 _ 1 1 _ 0 2 /I3 /0_I3 _ 1 S L in/p d a ta/1 A dm in istrato r F ri N ov 29 17 :41:53 200 2

ACTH

Insulin

Ubiquitin

*Cyto

chromeC

Ribonuc

leaseA

*Myoglobin*

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

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a.i.

/D = /da ta/N e w /d eri_ ca ra /29 _11 _02 /A 8/0_A 8 _1S Lin /pd ata /1 A dm in istrato r F ri N o v 29 17:25:4 3 2 002

CHCA/TFA/ACN DD

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/D = /data/N ew /deri_cara/29_11_02/A 14/0_A 14_1S Lin/pdata/1 A dm inistrator F ri N ov 29 17:29:53 2002

CHCA/TFA/ACN/OGP DD

4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 1 6 0 0 0 1 8 0 0 0m /z

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a .i.

/D = /d a ta /N e w /d e ri_ ca ra /2 9 _ 1 1 _ 0 2 /A 2 2 /0 _ A 2 2 _ 1 S L in /p d a ta /1 A d m in istra to r F ri N o v 2 9 1 7 :3 3 :0 9 2 0 0 2

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)

1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 m /z

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a .i.

/D = /d a ta /N e w /d e ri_ ca ra /2 1 _ 1 1 _ 0 2 /p e p m ix8 K 5 /0 _ K 5 _ 1 S L in /p d a ta /1 A d m in istra to r F ri N o v 2 2 1 2 :2 6 :3 6 2 0 0 2

SA/TFA/ACN SC

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/D = /da ta/N e w /d eri_ ca ra /13 _11 _02 /cara_K 6 /0_ K 6_ 1S Lin/p da ta/1 A d m inistrator T hu N o v 14 14:59:0 7 2 002

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/D = /da ta/N e w /d eri_ ca ra /13 _11 _02 /cara_K 6 /0_ K 6_ 1S Lin/p da ta/1 A d m inistrator T hu N o v 14 14:59:0 7 2 002

SA/TFA/ACN SC

1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 3 5 0 0 0 4 0 0 0 0 4 5 0 0 0 m /z

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a .i.

/D = /d a ta /N e w /d e ri_ ca ra /2 1 _ 1 1 _ 0 2 /p e p m ix8 K 1 0 /0 _ K 1 0 _ 1 S L in /p d a ta /1 A d m in istra to r F ri N o v 2 2 1 2 :3 8 :4 5 2 0 0 2

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/D = /data/N ew /deri_cara/21_11_02/pepm ix8K 10/0_K 10_1S Lin/pdata/1 A dm inistrator F ri N ov 22 12:38:45 2002

SA/TFA/ACN/OGP SC

1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 3 0 0 0 0 3 5 0 0 0 4 0 0 0 0 4 5 0 0 0 5 0 0 0 0 5 5 0 0 0 m /z

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a .i.

/D = /d a ta /N e w /d e ri_ ca ra /2 1 _ 1 1 _ 0 2 /p e p m ix8 K 1 1 /0 _ K 1 1 _ 1 S L in /p d a ta /1 A d m in istra to r F ri N o v 2 2 1 4 :2 2 :3 5 2 0 0 2

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a.i.

/D = /da ta/N e w /d eri_ ca ra /21 _11 _02 /pe pm ix8K 1 1/0_ K 11 _1S L in /pd ata /1 A dm inistrato r F ri N o v 22 14:2 2:3 5 2 002

SA/TFA/ACN/NC SC

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/D = /data/N ew /deri_cara/21_11_02/pepm ix8K 13/0_K 13_1S Lin/pdata/1 A dm inistrator F ri N ov 22 14:26:04 2002

SA/TFA/ACN/NC SC

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/D = /data/N ew /deri_cara/21_11_02/pepm ix8K 11/0_K 11_1S Lin/pdata/1 A dm inistrator F ri N ov 22 14:22:35 2002

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).

5749 5817 6756 8132 8596 8725 89949342 9818 9969 11741 13770 15167 1616516582 17461 18103 ~20600 21600 22642 ~24260

~27014 ~27830 ~28179 ~28840 ~29840 ~34660 ~36600~38890 ~41400 ~45700

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 KAmylaseProtoplasting mixture (Cellulase and Macerozyme)RNAse/DNAse

Range of characteristics – some totally resistant - some digested by proteinase and amylase

10 mm

Nanospray allows direct injection of single cell samples

Cell volumes samples > 10 – 100 plNanospraysSamples: > 100 – 200 nl

Dr Karim Al-Zubaidi. Single leaf cells – Tradescantia virginiana

(Proof of concept)

An obsession with micro capillaries ?

Single Cell Sampling and Analysis

SiCSA

Picolitre osmometry

X-ray microanalysis

Fluorescent microscopyImmunoassay

Capillary electrophoresis

/Data/bandat05/jun_05/20_jun_05/scsneg/2/pdata/1 Administrator Tue Oct 18 09:28:14 2005

Tradescantia SiCSA-ve ion. Nanospray50% Acetonitrile, 0.1% Triethylamine, 1% Chloroform

Sucrose ?

Sample from single Tradescantia leaf mesophyll cell (SiCSA)

Tradescantia epidermal cells

Ma

late

Ma

late

Ma

late

Ma

late

Glu

cose

Glu

cose

Glu

cose

Glu

cose

Su

cro

seS

ucr

ose

Su

cro

se

Citr

ate

Citr

ate

Citr

ate

Arg

Arg

inin

eA

rgin

ine

Arg

inin

e

Aco

nita

te ?

Aco

nita

te ?

Aco

nita

te ?

Aco

nita

te ?

Natalia Ivashikina