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

llightning@alquest.us

http://www.meetup.com/BayAreaLifeTech/

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