in vitro lc-ms approaches to deorphanization of human...

Zhongmei Tang

25 April 2010

Dr. F. P. Guengerich Lab

Department of Biochemistry &

Center in Molecular Toxicology

Vanderbilt University School of Medicine

In vitro LC-MS Approaches to Deorphanization of

Human Cytochrome P450 Enzymes

Why deorphanization of human P450 enzymes?

In vivoobservations

In vitroassays

Protein cDNA Gene

Traditional biochemistry

Developassay Purify Clone Sequence

PredictExpress

Determine substrates, relevance

21st century paradigm

Guengerich et al. Molecular Interventions 2010, in press.

Orphans: 2A7, 2S1,

2U1, 2W1, 3A43, 4A22,

4F11, 4F22, 4V2, 4X1,

4Z1, 20A1, 27C1

deorphanize

LC-MS based metabolomics approach

Vinayavekhin et al. ACS Chem. Biol. 2010, 5, 91-103.

• Commercial solutions

BlueFuse, MetAlign, MarkerLynx, etc.

• Freely available tools

MZmine, XCMS, DoGEX, etc.

Metabolite isolation

Detection

Data analysis

Discovery of General Endo- and Xenobiotics

(DoGEX)

Sanchez-Ponce et al. Anal. Chem. 2007, 79, 3355-3362.

RP450

16O2

R-16O

RP450

18O2

R-18O

Rno P450

RP450

16O2/18O2

R-16O

R

R-18O

1:1 1:1M/M+2

doublet

P450

substrate

16O2∕18O2

1:1

Activator

Substrate library: human tissue extract

The mixture was stirred for 20 min at 23 °C;

homogenate was filtered through paper

Tissue samples were homogenized in a CHCl3/CH3OH mixture

(2:1,v/v), final volume 20-fold that of the tissue sample

The crude extract was mixed with 0.2 volume of H2O

and centrifuged at 5000 rpm for 20 min at 23 °C

The lower organic layer was collected and

dried under N2 and stored at -70 °C

The dry extract was dissolved in 2 mL of ethanol

prior to incubation with P450

Strategy of my project: LC-MS metabolomic and isotopic labeling approach

Isotopic labeling incubation of P450 and tissue extract

LC-MS assay for metabolic profiling

Data analysis by DoGEX program

Characterization of candidate compounds

Tang et al. Anal. Chem. 2009, 81, 3071-3078.

Targeted substrate search for

P450 7A1 in human liver


HO OHO O

COOH

O

HOOC

O

OO O

Anhydrous Pyridine

65oC 6h

HOHO OH

( 16O/18O = 1:1 )

P450 7A1

16O2 / 18O2

1: 1

( 16O/18O = 1:1 )( 16O/18O = 1:1 )

Cholesterol 7 -OH cholesterol

Untargeted substrate searches for

P450s 1A2, 2C8, and 2C9 in human liver

1. Control incubations

2. LC-MS/MS, LIPIDMAPS

C14:0, C16:0, C16:1, C18:1,

C18:2, C20:4, and C22:6

1. TMS derivatization

2. GC-MS assayHydroxylation/epoxidation

products

1. In vitro incubation

(16O2∕18O2 =1:1)

2. LC-MS assay

3. DoGEX data analysis


Sterols XenobioticsFatty

acidsEicosanoids Vitamins Unknown

Putative

substratesExpression site

1B1 1A1 2J2 4F2 2R1 2A7

7A1 1A2 4A11 4F3 24A1 2S1 ? Skin7B1 2A6 4B1 4F8 26A1 2U1 Fatty acid

8B1 2A13 4F12 5A1 26B1 2W1 Carcinogens Cancer tissues

11A1 2B6 8A1 26C1 3A43

11B1 2C8 27B1 4A22

11B2 2C9 4F11Eicosanoids

& xenoboiticsLiver

17A1 2C18 4F22

19A1 2C19 4V2 ? Eye

21A2 2D6 4X1 Anandamide Brain

27A1 2E1 4Z1 Fatty acid Breast tumor

39A1 2F1 20A1 ? Liver

46A1 3A4 27C1 ? Liver

51A1 3A5

3A7

Human P450 groups based on their main substrates

Limited information regarding heterologous expression and functional

characterization:

• Being located in a cluster with the other five members of the P450 4F subfamily on

chromosome 19p13.1-2 and its mRNA being found mainly in human liver

(Cui X. et al., Genomics, 2000, 68, 161-166.)

• Being expressed in a yeast system (Saccharomyces cerevisiae) and Involving in

the metabolism of endogenous eicosanoids and some clinically active drugs

(Kalsotra A. et al., Toxicol. Appl. Pharmacol., 2004, 199, 295-304.)

• Being expressed insect cells (Spodoptera frugiperda) and kinetic studies on

oxidation of -hydroxy fatty acids.

(Dhar M. et al., J. Lipid Res., 2008, 49, 612-624.)

An untargeted metabolomic strategy as well as a more robust expression

system is required!

Human P450 4F11

P450 4F11 expression and purification

Table 1. Selection of N-terminal sequences of human P450 4F11 for heterologous expression. Amino acid changes are underlined.

Constructs N-terminal Amino Acid Sequences

4F11 native MPQLSLSWLGLGPVAASPWLLLLLVGGSWLLARVLAWTYTFYDNCRRLQCFPQPP

#1 2C3 rabbit MAKKTSSKGKLPQPP

#2 2C3 rabbit MAKKTSSKGKLQCFPQPP

#3 2E1 human MARQVHSSWNLQCFPQPP

#4 17A1 bovine MALLLAVFLAWTYTFYDNCRRLQCFPQPP

#5 2E1 human MARQVHSSWNLYDNCRRLQCFPQPP

#8 full length MAQLSLSWLGLGPVAASPWLLLLLVGGSWLLARVLAWTYTFYDNCRRLQCFPQPP

#11 17A1 bovine MALLLAVFLGPVAASPWLLLLLVGGSWLLARVLAWTYTFYDNCRRLQCFPQPP

• Bacteria: E. coli BL21-Gold(DE3)

• Plasmid: P450 4F11 bc construct #11

• Overnight culture (LB medium):

37 °C 250 rpm 16 h

• Large scale expression (TB medium):

37 °C 220 rpm 4 h before induction

(IPTG,5-ALA), 27 °C 190 rpm 40 h

• ~ 230 nmol P450 membrane was obtained

from 3-liter culture

Tang et al. Arch. Biochem. Biophys. 2010, 494, 86-93.

Table 2. Purification of human P450 4F11 from E. coli membrane fractions.

Purification step P450 (nmol) Yield (%)

Membrane 227 100

Solubilized fraction 214 94

Ni2+-NTA column fraction 106 47

HTP hydroxylapatite column

purified fraction

73 32

SDS-PAGE, Coomassie

stain

1,Solubilized fraction

2, Ni2+-NTA agarose-

purified fraction

3, Hydroxylapatite-

purified fraction

4, Protein Mr markers.

Aminopropyl SPE column procedure

Wash the column twice with 2 mL of hexane

Load the total extract (dissolve in 0.5 mL of CHCl3) on the column

Elute the column using 4 mL of CHCl3/2-propanol (2:1)

Elute the column using 4 mL of 2% acetic acid in diethyl ehter

Elute the column using 4 mL of CH3OH

Collect the three fractions and dried under N2 and store at -70 0C

Fraction1: neutral lipids

Fraction2:

fatty acids

Fraction3: phospholipids

Si

O

Si

O

OH

Si (CH2)3NH2

Kaluzny et al. J. Lipid Res. 1985, 26, 135-140.

Untargeted substrate search for

orphan P450 4F11 (liver extract fraction 2)

1. Control incubations

2. LC-MS/MS, LIPIDMAPS

3. Knowledge of components

of liver extract fraction 2

C16:0, C18:1,

C20:4, C22:6

1. TMS derivatization

2. GC-MS assay

-hydroxylation products

Tang et al. Arch. Biochem. Biophys. 2010, 494, 86-93.

Summary

LC-MS metabolomics and isotopic labeling approach has been developed and

validated by identifying 7 -OH cholesterol using P450 7A1 and liver extract.

The approach has been applied for untargeted substrate searches in liver extract

for known human P450 1A2, 2C8, and 2C9. Seven fatty acids have been identified

as the substrates.

The approach has been applied on true orphan P450s, four fatty acids have

been identified as the substrates of P450 4F11.

It is unclear if fatty acids are actually the main substrates for the

selected human P450s or whether they are just more readily detected

than other compounds during LC-MS assays and metabolomics?

Chemical derivatization is needed to improve the sensitivity!

Cl

SO O

N

Dansyl chloride

HN

R' Rprimary, secondary amines

OH

R

R OH

phenols

alcohol

Can dansyl chloride derivatize alcohols?

?

Representative compounds for dansylation

OH

H

O

H H

Testosterone

OH

H

O

H H

OH

6 -OH testosterone

HO

Vitamin D3 (Cholecalciferol)

OH

Cholesterol Vitamin A (Retinol)

HO

OHOH

O

Deoxycholic acid

OHHO

O

12-OH dodecanoic acid

OH

OOH

3-OH palmitic acid

OH

O

HOHO

O

Hydrocortisone Estrone (internal standard)

O

HO

H

H H

HO

H H

H H

Tang et al., in preparation

R OH+

Dansyl chloride

Cl

SO O

N

Dansylated alcohol

DMAP [(CH3)2CH]2NC2H5

in CH2Cl2

65 oC 1h

O

SO O

N

Alcohol

R

N

NE-A

DMAP DMAPN-acylpyridinium interdediate

N

NNuH

+

N

N+

A-E

B+ENu HB+A-

E = ElectrophileA = Counterion or its precursorNu = NucleophileNuH = SubstrateB = Strong base

Tang et al, in preparation

Reaction scheme for dansylation

R OH+

Dansyl chloride

Cl

SO O

N

Dansylated alcohol

DMAP [(CH3)2CH]2NC2H5

in CH2Cl2

65 oC 1h

O

SO O

N

Alcohol

R

N

NE-A

DMAP DMAPN-acylpyridinium interdediate

N

NNuH

+

N

N+

A-E

B+ENu HB+A-

E = ElectrophileA = Counterion or its precursorNu = NucleophileNuH = SubstrateB = Strong base

Procedure of dansylation reaction

Dry compound (5 nmol)

Diisopropylethylamine (2 L)

CH2Cl2 (100 L )

65 0C 1h, vortex mixing, centrifuged,

dried under N2, redissolved in CH3CN

Dansyl (1 mg )

DMAP (1 mg )

CH2Cl2(100 L )

1

2

LC-MS assayLC- Fluorescence

assay

LC-Radioactive

assay

Yield of dansylation

Cholesterol: 96%

Testosterone: 89%

O

SO O

N

R

m/z 252

Re

lative

Ab

un

da

nce

Time (min)

0 2 4 6 8 10 12 14 160

50

1000

50

1000

50

1000

50

1000

50

1000

50

1000

50

100 5.30

5.07

10.06

5.95

4.81

9.80

4.806.06

7.86

3.16E5

2.37E7

3.64E5

3.54E5

3.71E5

1.33E5

2.37E5

0 2 4 6 8 10 12 14 160

50

1000

50

1000

50

1000

50

1000

50

1000

50

1000

50

100 5.30

3.56

5.98

6.07

7.01

6.61

7.60

3.16E5

1.01E7

3.33E6

3.64E5

3.69E4

4.41E4

9.45E4

m/z 504 171

m/z 522 252

m/z 620 252

m/z 520 252

m/z 618 252

m/z 450 252

m/z 506 252 m/z 355 252

m/z 596 252

m/z 430 252

m/z 626 252

m/z 386 252

m/z 538 252

m/z 504 171

Testosterone

Cholesterol

Vitamin A

Estrone

Vitamin D3

12-OH dodecanoic acid

3-OH palmitic acid

Estrone

6 -OH testosterone

6 -OH testosterone-2

Deoxycholic acid

Deoxycholic acid-2

Hydrocortisone

Hydrocortisone-3

40 eV

25 eV

22 eV

25 eV

35 eV

15 eV

25 eV

40 eV

25 eV

15 eV

15 eV

15 eV

25 eV

15 eV


The mixture of the above compounds was used for dansylation reaction. LC-MS analysis of dansylated compounds

using MRM under ESI (+): 50 pmol of each compound and 1 pmol of estrone (I.S.) were injected.

Dansylation application

1. Identifying P450 reaction products in tissue extract using LC-MS

Method 1: 18O2/16O2 system and DoGEX program, full scan, ESI+

Method 2: Differential analysis (wt vs ko, or ± enzyme), parent ion scan (m/z 252)

2. Measuring enzyme activity (e.g., 7A1+ cholesterol, 3A4+testosterone)

3. Quantifying representative compounds in biological samples

(e.g., cholesterol in human liver)


E.g., 7A1 + human liver extract

Signal was increased by 103-fold!

Improved strategy for new substrate search

Isotopic labeling incubation of P450 and tissue extract

LC-MS assay for metabolic profiling

Data analysis by DoGEX program

Characterization of candidate compounds

dansylation

New substrates!!!

Acknowledgements

Dr. F.P Guengerich

Martha Martin

Giovanna Salamanca

Yi Xiao

Goutam Chowdhury

Zhenhua Tian

Raku Shinkyo

Christal D. Sohl

Dr. Guengerich Lab

Dr. Brash Lab

Dr. Alan Brash

William Boeglin

Grant

NIH grants R37 CA090426

and P30 ES000267

MS research centerDr. David Hachey

Dr. M. Wade Calcutt

in vitro lc-ms approaches to deorphanization of human...

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