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