Mass spectrometric analysis of cyp450s following mechanism based inactivation Lightning 2012
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Transcript of Mass spectrometric analysis of cyp450s following mechanism based inactivation Lightning 2012
Mass Spectrometric Analysis of CYP450s Following Mechanism-Based Inactivation
Luke Lightning, PhDAlquest Therapeutics
San Francisco, CA
Genentech Presentation7/11/2012
Outline
• Brief Introduction• LC/MS Analysis of Intact CYP450s• Identification of Peptide Adducts• Other Studies• Conclusions and Future Directions
Known Pathways of CYP450 Mechanism-Based Inactivation
NN
NN
NN
NN
MBI*
MBI
Fe
Fe
FeN
N
NN
Fe
NN
N N
MBI*
Cys
MM 1A2 2A6 2C9 2C9 3A4 C175R
• Charge States = 25 • Predicted MM = 55,575.1 Da• Error = 0.006% (3.5 Da)
P450 2C9
Purification and Mass Spectral Analysis of P450s
• specific contents = 14.3-16.6 nmol/mg
ESI-MS
kDa
94
67
43
30
20
55,578.6 Da
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)
• a
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
CYP450 2B1 + 8-MOP: HPLC Separation
0
50
100
150
200Absorbance (214 nm)
CYP450 reductase CYP2B1
b5, Heme
250
Radioactivity (dpm) 200
100
300
400
Binding Stoichiometry = 0.7 ± 0.1
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)
Time (min) 8.00
Biochemistry 37, 13184-13193 (1998)
Methods in Enzymology, Volume 357, page 296-300 (2002)
Mechanism-based Inactivators of CYP450s
NO
N
N N
NH
OH OHH
O O
8-MOP
OCH 3
OO O
L-754,394
Tienilic Acid
SO
OCH2COOHCl
Cl
X
O
2A6 & 2B1
2C9
3A4
Nu:CYP450
CYP2C9
Tienilic Acid
+
2C9 Diadduct
Monoadduct
SO
OCH2COOHCl
Cl
monoadduct2C9
(+) GSH
CYP2C9 + Tienilic Acid
55,578.6 Da
(+) 344 and (+) 694 Da correlate w/ addition of TA-OH to 2C9 suggested thiophene epoxide mechanism is operative
(+) 694 ± 4
(+) 344 ± 1
Biochemistry 38, 2312-2319 (1999)
CYP2B1 + 8-MOP: LC/MS Analysis
55,925.7 Da
56,163.6 Da
+ 8-MOP
CYP2B1 CYP2B1/8-MOP + CYP2B1
• = 237.9 Da (furanoepoxide = 234.2 Da)• similar results obtained with CYP450 2A6
• predicted = 56,933.8 Da• error = 8.1 Da (0.01%)
CYP2B1
Biochemistry 37, 10047-10061 (1998)
Methods in Enzymology, Volume 357, page 296-300 (2002)
2.5
kDa200
11697
3.5
6655
3731
14
2217
6
1114
8
kDa
MM 2B1 + MM 8-MOP
CYP2B1-8-MOP
Radioactivity (dpm)
CNBr Peptide
4030201000
200
400
600
800
Time (min)
CNBr
8-MOP
NN
NN Fe
Radioactivity
MALDI-MS
MALDI-MS: CYP2B1 + 8-MOP
Counts
35000
30000
25000
20000
15000
100005000
0
Mass (m/z)
2500 2600 2700 2800 2900 3000
2722.1
2721.9 ± 0.1 290-ISLLSLFFAGTETSSTTLRYGFLLM-314 (2721.2 Da)
SRS-4 (I helix)
CYP2B1 + 8-MOP
8-MOP
CYP3A4 + L-754,394
SDS-PAGE
HPLC MALDI-MSBiochemistry 39, 4276-4287
(1999)
4704.2 ± 1.0
Counts
Mass (m/z)
3000
2000
1000
0
4000 4600 4900 5200 55004300
4705.2
4000
275-IDSQNSKETESHKALSDLELVAQSIIFIFAGYETTSSVLSFIM-317 (4703.2 Da)
SRS-4 (I-helix)
MALDI-MS: CYP3A4 + L-754,394
CYP3A4 + Raloxifene
raloxifene-adducted peptide, 237-NICVFPR-243.
No radiolabel
adductedpeptide
fromproteinase K
digest
PIA adducted CYP3A4 (Cys-reacting)
MS2
MS3
Cys239
3
Tyrosine 75
Cys239
peptide from tryptic digest
2011
CYP2B6
CYP2B6 + clopidogrel
CYP2B6+ clopidogrel+
DTT
2011
2006
2005
2004Other studies
Other studies2010
2011
2012
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
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
Future Directions – MALDI?
• Tumor specific protein signals were detected
• Proteomic information was extracted
• Try with HLMs
Thank you!!
Luke Lightning, PhD
Alquest Therapeutics
http://www.meetup.com/BayAreaLifeTech/
Next event: Happy Hour in SF, Thursday 7/12/2012