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.
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