INSTRUMENTATION II

BIOCHEMICAL APPLICATIONS IN MIGRAINE RESEARCH

Alfred Nji Fonteh, PhD.HMRI- Molecular Neurology Program

99 N El Molino AvenuePasadena, CA 91101

OBJECTIVES

•Overview of hypotheses •Important molecules in migraine research•Methods for measuring fatty acids and eicosanoids

•Research data•Methods for measuring steroid hormones

•Research data•Methods for measuring protein and peptides

•Research data•Proposed pathway from discovery to the consumer

A) COMPOSITIONAL ANALYSES •DNA•PROTEINS•LIPIDS•CARBOHYDRATES•ELEMENTS

B) MECHANISM BASED ANALYSES •VASOACTIVITY (peptides, fatty acids, hormones, NT)•INFLAMMATION (prostanoids, peptides, cytokines)•PAIN (receptors, fatty acid amides, peptides)•OXIDATION (fatty acids, proteins)•ION CHANNELS (receptors, elements, NT)

Free Fatty Acids

LC-APCI-ECNICI-MS/MS

• Add IS to CSF• Extract steroids• Derivatize to PFBr• Run on C8 LC using

methanol gradient• Detect ions using

ECNI-MS/MS

EXTRACT LIPIDS

(Base hydrolysis for total)

DERIVATIZEPFBr + DIPE

Add IS

SPE

NICI-GC/MSLC-NICI/APCI-MS

Analyze Data

Total Fatty Acids

RT: 0.00 - 35.01

0 5 10 15 20 25 30 35

Time (min)

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

300000000

320000000

340000000

360000000

380000000

Inte

nsity

RT: 17.63AA: 5839780484AH: 373093564

RT: 19.03AA: 1264205886AH: 141080013

RT: 14.25AA: 1329574443AH: 88186711

RT: 3.52AA: 71867784AH: 3288126

RT: 12.13AA: 109978770AH: 3175908

RT: 24.22AA: 3752123AH: 300232

RT: 31.53AA: 455204AH: 158252

NL:3.91E8

TIC MS ICIS Fatty acid Mix

Fatty acid Mix RT: 0.01 - 35.01 Mass: 250 - 335 NL: 9.03E7

5 10 15 20 25 30 35

Time (min)

250

260

270

280

290

300

310

320

330

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

90000000

Inte

nsity

RT: 0.00 - 35.01

0 5 10 15 20 25 30 35

Time (min)

0

20000000

40000000

60000000

0

20000000

40000000

0

20000000

40000000

Inte

nsity

0

20000000

40000000

60000000

RT: 17.65AA: 751136820AH: 65092763

RT: 12.54AA: 23764959AH: 1079715

RT: 19.44AA: 1297077AH: 113886

RT: 17.59AA: 553492826AH: 49632925

RT: 3.44AA: 47982397AH: 1685782

RT: 19.88AA: 9238832AH: 1105248

RT: 24.15AA: 946842AH: 80694

RT: 13.97AA: 1125715AH: 75826

RT: 16.93AA: 696528372AH: 54238331

RT: 18.53AA: 2568120AH: 253293

RT: 10.92AA: 4435371AH: 157023

RT: 16.21AA: 1610002AH: 139497

RT: 1.82AA: 2064766AH: 99920

RT: 17.53AA: 774130886AH: 68686774

RT: 18.50AA: 613636AH: 91001

RT: 16.45AA: 412095AH: 43369

NL:6.54E7

m/z= 303.02-304.02 MS ICIS Fatty acid Mix

NL:4.98E7

m/z= 310.50-311.50 MS ICIS Fatty acid Mix

NL:5.45E7

m/z= 301.02-302.02 MS ICIS Fatty acid Mix

NL:6.89E7

m/z= 327.10-328.10 MS ICIS Fatty acid Mix

RT: 0.00 - 35.01

0 5 10 15 20 25 30 35

Time (min)

0

50000000

100000000

0

50000000

0

50000000

100000000

Inte

nsity

0

10000000

0

50000000

RT: 21.37AA: 1290134084AH: 88899855

RT: 14.25AA: 1083612614AH: 72624978

RT: 9.30AA: 62116009AH: 2022674

RT: 23.04AA: 3275400AH: 348623

RT: 2.18AA: 3647841AH: 196539

RT: 18.53AA: 172449932AH: 16701739

RT: 6.17AA: 5827783AH: 142458

RT: 10.56AA: 1536296AH: 72418

RT: 19.03AA: 1061372331AH: 116497519

RT: 12.03AA: 77147972AH: 2677121

RT: 17.65AA: 798498390AH: 71892364

RT: 3.51AA: 30716182AH: 1115946

RT: 8.95AA: 3958675AH: 120579

RT: 18.93AA: 448832AH: 84763

RT: 18.36AA: 1111443559AH: 106626685

RT: 10.42AA: 36919333AH: 1281715

RT: 21.62AA: 1874966AH: 189448

RT: 1.37AA: 2241145AH: 163658

NL:8.91E7

m/z= 254.93-255.93 MS ICIS Fatty acid Mix

NL:1.67E7

m/z= 270.00-271.00 MS ICIS Fatty acid Mix

NL:1.17E8

m/z= 282.98-283.98 MS ICIS Fatty acid Mix

NL:7.21E7

m/z= 278.90-279.90 MS ICIS Fatty acid Mix

NL:1.07E8

m/z= 280.98-281.98 MS ICIS Fatty acid Mix

Control Migraineur (-HA)

Migraineur (+HA)

0

1

2

3EPA/DGLA/LA

Fat

ty a

cid

s (n

mol

/nm

ol li

pid

ph

osp

hor

us)

Control Migraineur (-HA)

Migraineur (+HA)

0.0

0.1

0.2

0.3

0.4

0.5

AA/LNA -Ist BarDHA -2nd Bar

Control Migraineur (-HA)

Migraineur (+HA)

0.0

0.2

0.4

0.6

0.8 OA/SA

Control Migraineur (-HA)

Migraineur (+HA)

0

10

20

30

40

50

60Total FA

PUFA levels in CSF

T XA2

T XB2

Arachidonic AcidGC/MS, HPLC/MS/MS

15 HETE

12 HETE

5-HETE

ELISA, GC/MS, HPLC/MS/MS

ELISA, GC/MS, HPLC/MS/MS PGG2

GC/MS, HPLC/MS/MS

PGH2HPLC/MS/MS PGI2

6-keto-PGF1aELISA, GC/MS

2,3-dinor-6keto-PGF1aGC/MS

PGE2ELISA, GC/MS, HPLC/MS/MS

PGA2 GC/MS, HPLC/MS/MS

15-keto-dihydro-PGE2 GC/MS, HPLC/MS/MS

LTA4

LTC4ELISA, HPLC/MS/MS

LTB4ELISA, GC/MS, HPLC/MS/MS

LTD4HPLC/MS/MS

LTE4HPLC/MS/MS

PGF2aELISA, GC/MS, HPLC/MS/MS

15-keto-13,14-dihydro-PGF2aGC/MS, HPLC/MS/MS

ELISA, GC/MS, HPLC/MS/MS

PGD2ELISA, GC/MS, HPLC/MS/MS

PGJ2ELISA, GC/MS, HPLC/MS/MS

2,3-dinor-TXB2ELISA, GC/MS

ANALYTICAL METHODS IN EICOSANOID RESEARCH

Prostaglandin H2 Synthase

membrane binding domain

ibuprofen analog

heme

PDB 1PGE

EICOSANOID MEASUREMENT•EIA/RIA/ELISA

•BIOASSAYS•TLC/HPLC

•NICI-GC/MS

DERIVATIZE (methoxyime-pentafluorobenzylester trimethylsilyl esters)

1) Methoxylamine (MOX for ketone group)

2) 20 % pentafluorobenzyl bromide (PFBr) and 20 % diisopropylethylamine(DIPE) in acetonitrile for carboxyle groups)

3) N,O-bis(trimethyl-silyl)-trifluoroacetamide (BSTFA) for hydroxyl groups

EXTRACT LIPIDS(add 4-5 vols of ethanol)

Add IS SPE

NICI-GC/MS + ANALYSIS OF DATA

TOTAL ION CHROMATOGRAM