Mass spectrometric analysis of cyp450s following mechanism based inactivation Lightning 2012
1. Mass Spectrometric Analysis of CYP450s Following
Mechanism-Based Inactivation
Luke Lightning, PhD
Alquest Therapeutics
San Francisco, CA
Genentech Presentation
7/11/2012
2. Outline
• Brief Introduction
• LC/MS Analysis of Intact CYP450s
• Identification of Peptide Adducts
• Other Studies
• Conclusions and Future Directions
3. Known Pathways of CYP450 Mechanism-Based Inactivation
MBI*
N
Fe N
N
N
Fe
MBI
MBI*
N
N N N
Fe N Fe N
N N N N
N N Cys
4. Purification and Mass Spectral Analysis of P450s
MM 1A2 2A6 2C9 2C9 3A4 P450 2C9
C175R
kDa
55,578.6 Da
94
67
ESI-MS
43
30
20
• specific contents = 14.3-16.6 nmol/mg • Charge States = 25
• Predicted MM = 55,575.1 Da
• Error = 0.006% (3.5 Da)
5. Standard Inactivation Experimental Protocol
• CYP450:reductase:cytochrome b5 (various ratios, 1 nmol CYP450)
• 50 mM potassium phosphate buffer
• Dialyze overnight at 4ºC to remove glycerol and/or detergent
• 50 µg DLPC, 2000 U/mL catalase added
• Set on ice for 60 min
• Preincubate at 30ºC for 2 min with inactivator (1% MeOH maximum)
• Initiate reaction with NADPH and incubate for 60 min
• Set on ice until LC/MS/MS analysis of intact protein or incubate with
protease or CNBr (reaction times vary) for peptide digests
• Can pre-treat the intact protein mixture with denaturants (e.g. Guanidinum
HCl, urea)
6. Standard LC/MS/MS Analysis
• HPLC column: Poros R2 perfusion column (4.6 x 100 mm) from Applied
Biosystems (Cambridge, MA)
• Buffer A – 0.05% TFA (pH 3.0), Buffer B – 0.05% TFA in 95:5% ACN:H2O
• Flow rate: 3 mL/min with 50 µL diverted to the MS
• 300 pmol of spectrally detectable CYP450 or 1 nmol of inactivated CYP450
and components was injected onto the system
• VG Quattro II triple quad ESI-MS running MassLynx software
• Data acquisition from m/z 200-2000Da
• Improvements with time:
– Can purchase purified CYP450s, CYP450 reductase, and cytochrome b5
– Mass spectrometers and software (e.g. SEQUEST)
– 2.1 x 30 mm columns lower flow rates, less protein required
7. CYP450 2B1 + 8-MOP: HPLC Separation
250
200 CYP450 reductase CYP2B1
Absorbance
150
(214 nm)
100
50
b5, Heme
0
0 Time (min) 8.0
400
300 Binding Stoichiometry
Radioactivity
(dpm) = 0.7 ± 0.1
200
100
8-MOP binding is specific for the apoprotein of 2B1
short HPLC run time (8-10 min)
hydrolysis of the label occurs (e.g. HPLC, SDS-PAGE, & enzymatic digest)
Biochemistry 37, 13184-13193 (1998)
9. Mechanism-based Inactivators of CYP450s
8-MOP 2A6 & 2B1
O O O
OCH3
O
S
X
2C9
Tienilic Acid Cl O
OCH2COOH
Cl
Nu:
CYP450
N OH OH 3A4
L-754,394 H
O N N
N
O O
NH
10. CYP2C9 + Tienilic Acid
Monoadduct
55,578.6 Da (+) 344 ± 1
2C9 Diadduct
CYP2C9 (+) 694 ± 4
+ (+) GSH
2C9 monoadduct
O
S
Tienilic Acid
Cl OCH2COOH
Cl
(+) 344 and (+) 694 Da correlate w/ addition of TA-OH to 2C9
suggested thiophene epoxide mechanism is operative
Biochemistry 38, 2312-2319 (1999)
11. CYP2B1 + 8-MOP: LC/MS Analysis
CYP2B1 CYP2B1/8-MOP + CYP2B1
56,163.6 Da
55,925.7 Da
CYP2B1
+ 8-MOP
• predicted = 56,933.8 Da • = 237.9 Da (furanoepoxide = 234.2 Da)
• error = 8.1 Da (0.01%) • similar results obtained with CYP450 2A6
Biochemistry 37, 10047-10061 (1998)
19. raloxifene-
adducted
PIA adducted CYP3A4
peptide,
(Cys-reacting) 237-NICVFPR-243.
Cys239
No radiolabel MS2
adducted
peptide
from
proteinase K
digest
MS3
20. Cys239
3
peptide from tryptic digest
Tyrosine 75
24. Conclusions
• LC/MS analyses of intact CYP450s is possible
– Short run times, accurate
• Adducted intact proteins and peptide adducts can be
identified without use of a radiolabeled molecule
25. Future Directions?
• Crystal structures of adducted proteins
• Proteomic analysis and scanning for adducts?
– Microsomes, hepatocytes
– Supersomes
– Labeled compounds or isotope labeled proteins
– Affinity purification techniques
– Potential issues with ESI
• Amount of protein required clogging of columns
• Stability of adducts to workup conditions
– MALDI
26. Future Directions – MALDI?
• Tumor specific protein signals
were detected
• Proteomic information was
extracted
• Try with HLMs
27. Thank you!!
Luke Lightning, PhD
Alquest Therapeutics
llightning@alquest.us
http://www.meetup.com/BayAreaLifeTech/
Next event: Happy Hour in SF, Thursday 7/12/2012