Quantitative Protein Analysis of CYP450 Induction via LC-MRM Analysis

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Cytochrome P450 Proteins

− Cytochrome P450 enzymes are mainly expressed in liver and are responsible for oxidative metabolism of drugs, environmental pollutants, carcinogens, etc

− Cytochrome P450 Family of Enzymes – 70 p450 protein families in humans– Over 200 different subfamilies / isoforms– Each isoform has different substrate specificities, varied inducibility by

different drugs

− Important in drug development– Changes in expression of specific isoforms provide information on toxicity

of different drugs – Individual patient basal expression levels affect responsiveness to drugs

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Human CYP EnzymesDrug Metabolism

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Current Methodologies for Assessing CYP450 Induction− mRNA techniques

– Measure the amount of messenger RNA expressed for each enzyme isoform– Assesses only CYP induction from gene transcription changes

− Enzymatic activity – Induction by quantifying the metabolite of a CYP-specific probe substrate

generated from treated hepatocytes– Relies on the specificity of enzymatic conversion of probe substrates, and a

different probe substrate is required for each CYP isozyme which is not always possible

− Western Blotting techniques– Measures the actual protein levels of CYP450 enzymes using isoform-specific

antibodies. Currently there are only a few good antibodies that are isoform specific

Major Challenges in Induction Assay:

Assay selectivity, and sensitivity; Different technical expertise and equipment are needed for mRNA or Western Blotting assessment

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Challenges of Current Approaches

− Western Blot Analysis of p450 Expression– Using commercially available

antibodies to the various subfamilies of P450 proteins, Western Blot analysis can be used to monitor changes in protein expression

– However, commercial antibodies are not available for all protein isoforms

– Some antibodies recognize multiple isoforms

− An MS-based approach could provide sensitivity and specificity through the detection of individual peptides from specific P450 isoforms

Cyp1a1

Cyp1a2

Cyp2e1

Cyp3a4

Co

ntr

ol

PB

in

du

ce

d

Changes in expression in response to treatment with phenobarbitol

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CYP Induction Assay: LC-MS/MS Solution

− An LC-MS/MS-based approach can provide sensitivity and specificity through the detection of individual peptides from specific CYP450 isoforms

− A fast MS scan speed and the Scheduled MRM™ Algorithm allows for multiplexed protein quantitation

− A CYP450 protein assay kit including all reagents, sample preparation procedure and established LC/MS/MS conditions provides easy protein quantitation for human induction studies using current DMPK resources

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Multiple Reaction Monitoring (MRM)

− Highest specificity and sensitivity for detecting components in a complex mixture

− Requires QTRAP® System or triple quadrupole MS capability

− Largest linear dynamic range for quantitation

− Well accepted as the MS technique for quantitation (Pharmaceutical Industry)

Fragment peptide

Select Peptide Select Fragment

Fragmentation Cell

Detect Fragment

Mass Analyzer

Mass Analyzer

Detector

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Sample Preparation for LC/MS/MS Analysis of Protein Therapeutics− Problem: Protein therapeutics and larger peptide therapeutics are

typically too large to directly quantitate using standard MRM assays in mass spectrometry

− Solution: Enzymatically digest the protein or large peptide therapeutic into small peptides and monitor one or more peptides as a surrogate– Trypsin is the enzyme of choice for several reasons:

– Tryptic peptides are a good size for MRM assays (not too large)

– Tryptic peptides tend to fragment well leading to good MRM assays

– Trypsin digest quality can be very good when a high grade of trypsin is used

Protein

Enzyme (Trypsi

n)

Peptides

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Quantifying Proteins by Multiple Reaction Monitoring

Intact protein

Enzyme - Trypsin

Peptide fragments

200 400 600 800m/z

0

MS/MS – Q3 m/z

**

**

Peptide Q1 m/z

Stable Isotope Labeled Internal Standards

MRM Method MRM Results

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General Strategy for Protein Quant using SIS Peptides

Generate a list of peptides that uniquely

identify each p450 isoform.

Synthesize each of these peptides with a

heavy amino acids

MRM LC/MS/MS

Sequence of Target Protein in sample

Design MRM method – monitor heavy and

light peptides

RQLYSLVGITK*

KLQISSDVLAR*RYILNDAVEIR*

…KLQISSDVLAR…

H2N

COOH

..RQLYSLVGITK..…RYILNDAVEIR…

Internal Std

Target

= Concentration of target protein

Area of target

Area of Internal std

* [ Int. Std. ]

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ControlMicrosomes

Trypsin digest

1D LC-MRM20 min run

ReduceAlkylate

Concentration of control P450

Concentration of induced P450

Synthetic heavy peptides-representative of each P450 studied

-for internal standard and concentration curve

Mix Mix

InducedMicrosomes

1D LC-MRM20 min run

Trypsin digest

ReduceAlkylate

[Peak Area Smp/Peak Area Std] *CStd

[Peak Area Smp/Peak Area Std] *CStd

P450 Peptide Assay Workflow

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Scheduled MRM™ AlgorithmImproving MRM Method Efficiency by Maximizing Analyte Utilization

− Each MRM monitored only across its expected elution time

− concurrent MRMs

− Maintain cycle time and dwell time

− effective duty cycle for every peptide

− Maintain analytical precision

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LC-MRM Assay of CYP Proteins

− High assay robustness through monitoring – Multiple

MRMs per peptide

– Multiple peptides per protein

3A4 -Pep 1

3A4 -Pep 2

3A4 -Pep 3

2B6 -Pep 1

2B6 -Pep 2

2B6 -Pep 31A2 -Pep 1

1A2 -Pep 2

1A2 -Pep 3

3A5 -Pep 3

3A5 -Pep 1

3A5 -Pep 2

CYP 1A2

CYP 2B6

CYP 3A4

CYP 3A5

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How Consistent are MRMs to each Peptide

2B6 Peptide 3

0

5

10

15

20

25

30

35

1mg/mL 1mg/mL 1mg/mL 2mg/mL 2mg/mL 2mg/mL 3mg/mL 3mg/mL 3mg/mL

fmo

l o

n c

olu

mn

MRM 1

MRM 2

MRM 3

Sample 1 Sample 2 Sample 3

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Peptide Consistency for CYP 1A2

0

20

40

60

80

100

120

140

160

1mg/mL 2mg/mL 3 mg/mL

fmo

l on

co

lum

n

Peptide 1

Peptide 2

Peptide 3

Sample 1 Sample 2 Sample 3

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Typical Western Blot Data from Induction Study

− The typical results seen in Western blot analysis of protein expression correlates with the observed LC/MS results

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XIC of (a) control and (b) 3-MC induced microsomes for the one of the peptides from Cyp1A2.

(a) (b)Control Sample 3-MC Induced Sample

Sample

Sample

StandardStandard

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LC-MS/MS ProteinAll Cytochrome P450 Proteins

− Adding CYP3A5 data relative to other CYPs

– Protein expression changes illustrate 3A5 is inducible

– 3-MC – minimal induction of 3A5

– (PB) – Significant induction of CYP3A5

– (RIF) – Small induction CYP3A5

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Conclusions - CYP Induction AssayLC-MS/MS Protein Expression Analysis

− Highly sensitive, specific, and fast Multiple Reaction Monitoring (MRM) method has been developed: – 12 different peptides representing 4 unique P450 proteins (CYP 1A2,

2B6, 3A4 and 3A5) were simultaneously monitored and quantified

– 2B6, a lower abundant CYP, is easily detected showing good dynamic range of method

− Largest protein expression change was observed for microsomes prepared from RIF induced hepatocytes – Cyp3A4 showed an increase in expression upon drug treatment of ~50-fold over control. – S9 or microsomal subcellular fractions can be used

− This method was in excellent agreement with existing methods (mRNA, enzyme activity assays)

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Human Induction Kit (100 Assays)Starter Kit Contents

− Heavy peptide mix

− Denaturant, Reducing reagent, Alkylating reagent

− Digestion buffer

− Trypsin

− Peptide column

− Acquisitions methods for– AB SCIEX Triple Quad™ 5500 and QTRAP® 5500 systems– API 4000™ system, 4000 QTRAP® system, API 5000™ system

− Quantitation methods for MultiQuant™ software 1.2

− Microsoft Excel 2007 results template

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Acknowledgements

− AB SCIEX– Sean Seymour

– Christie Hunter

– Lydia Nuwaysir

− CellzDirect– Jeanette Hill

– Rob Taylor

Thank You for your Attention

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Trademarks/Licensing

− For Research Use Only. Not for use in diagnostic procedures.

− The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.  AB SCIEX™ is being used under license.

− © 2010 AB SCIEX. All rights reserved. Information subject to change without notice.