U.S. patent application number 14/465460 was filed with the patent office on 2015-02-12 for mass spectrometry quantitation of p450 protein isoforms in hepatocytes.
The applicant listed for this patent is DH Technologies Development Pte. Ltd.. Invention is credited to Subhasish Purkayastha, Brian L. Williamson.
Application Number | 20150045256 14/465460 |
Document ID | / |
Family ID | 43876537 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150045256 |
Kind Code |
A1 |
Williamson; Brian L. ; et
al. |
February 12, 2015 |
MASS SPECTROMETRY QUANTITATION OF P450 PROTEIN ISOFORMS IN
HEPATOCYTES
Abstract
A method for screening a drug for cytochrome P450 (CYP)
induction is provided and can include incubating the drug with a
microsome-containing biological sample and then quantitating at
least one cytochrome P450 isoform. The isoforms can be selected
from 2B6, 3A4, 1A2, and 3A5 isoforms. In some embodiments, the
method uses liquid chromatography tandem mass spectrometry
(LC-MSMS). A quantitated value can be compared to a threshold value
and the drug can be determined to exhibit an acceptable CYP
induction potential when the quantitated value does not exceed the
threshold value. Isolated peptides are also provided.
Inventors: |
Williamson; Brian L.;
(Ashland, MA) ; Purkayastha; Subhasish; (Acton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DH Technologies Development Pte. Ltd. |
Singapore |
|
SG |
|
|
Family ID: |
43876537 |
Appl. No.: |
14/465460 |
Filed: |
August 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12904520 |
Oct 14, 2010 |
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14465460 |
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61252648 |
Oct 17, 2009 |
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61252430 |
Oct 16, 2009 |
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Current U.S.
Class: |
506/12 ;
435/25 |
Current CPC
Class: |
G01N 2560/00 20130101;
G01N 33/6848 20130101; G01N 2500/20 20130101; G01N 30/7233
20130101; G01N 2500/10 20130101; G01N 33/5023 20130101; C12N 9/0077
20130101; C12Q 1/26 20130101; G01N 2500/04 20130101; Y10T 436/24
20150115; G01N 2030/8831 20130101; G01N 33/502 20130101; G01N
33/5067 20130101; G01N 2333/90245 20130101 |
Class at
Publication: |
506/12 ;
435/25 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/50 20060101 G01N033/50 |
Claims
1. A method for determining an amount of at least one isoform of
cytochrome P450 (CYP) in a sample, said method comprising
subjecting said sample to an analysis with a liquid chromatograph
tandem mass spectrometer system, said system comprising a triple
quadrupole instrument and Multiple Reaction Monitoring (MRM), said
analysis comprising detecting at least one isolated peptide of CYP
3A4, CYP 1A2 or CYP 3A5.
2. The method of claim 1 wherein the inform is CYP 3A4 and said
isolated peptide has the amino acid sequence of SEQ ID NO: 5, SEQ
ID NO: 6, or SEQ ID NO: 7
3. The method of claim 2, wherein the amino acid sequence is SEQ ID
NO:5 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
440/549, 440/650, or 440/532 in said system and processing data
from said monitoring to determine said amount.
4. The method of claim 2, wherein the amino acid sequence is SEQ ID
NO:6 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
704/794, 704/929, 564/689, 564/745, or 564/790 in said system and
processing data from said monitoring to determine said amount.
5. The method of claim 2, wherein the amino acid sequence is SEQ ID
NO:7 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
798/819, 798/932, or 798/1004 in said system and processing data
from said monitoring to determine said amount.
6. The method of claim 1 wherein the isoform is CYP 1A2 and said
isolated peptide has the amino acid sequence of SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:
13 or SEQ ID NO: 14.
7. The method of claim 6, wherein the amino acid sequence is SEQ ID
NO:8 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
432/636, 432/535, or 432/478 in said system and processing data
from said monitoring to determine said amount.
8. The method of claim 6, wherein the amino acid sequence is SEQ ID
NO:9 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
482/800, 482/628, or 482/743 in said system and processing data
from said monitoring to determine said amount.
9. The method of claim 6, wherein the amino acid sequence is SEQ ID
NO:10 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
491/721, 491/834, or 491/535 in said system and processing data
from said monitoring to determine said amount.
10. The method of claim 6, wherein the amino acid sequence is SEQ
ID NO:11 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
528/501, 528/614, or 528/727 in said system and processing data
from said monitoring to determine said amount.
11. The method of claim 6, wherein the amino acid sequence is SEQ
ID NO:12 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
571/783, 571/971, 571/1028, 381/587, 381/474, or 381/375 in said
system and processing data from said monitoring to determine said
amount.
12. The method of claim 6, wherein the amino acid sequence is SEQ
ID NO:13 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
695/695, 695/837, or 695/950 in said system and processing data
from said monitoring to determine said amount.
13. The method of claim 6, wherein the amino acid sequence is SEQ
ID NO:14 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
536/795, 536/584, or 536/698 in said system and processing data
from said monitoring to determine said amount.
14. The method of claim 1 wherein the isoform is CYP 3A5 and said
isolated peptide has the amino acid sequence of SEQ ID NO: 15, SEQ
ID NO 16, or SEQ ID NO: 17
15. The method of claim 14, wherein the amino acid sequence is SEQ
ID NO:15 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
468/581, 468/679, or 468/736 in said system and processing data
from said monitoring to determine said amount.
16. The method of claim 14, wherein the amino acid sequence is SEQ
ID NO:16 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
470/494, 470/608, or 470/722 in said system and processing data
from said monitoring to determine said amount.
17. The method of claim 14, wherein the amino acid sequence is SEQ
ID NO:17 and said analysis is performed by monitoring a
precursor-product ion pair transition having an m/z value of about
589/747, 589/696, or 589/647 in said system and processing data
from said monitoring to determine said amount.
18. A method for screening a drug for cytochrome P450 (CYP)
induction, comprising: incubating the drug with a
microsome-containing biological sample; determining a quantitated
value of at least one of a CYP 3A4, CYP 1A2 or CYP 3A5 isoform in
said drug incubated with the microsome-containing biological
sample, said determining comprising, subjecting said drug incubated
with the microsome-containing biological sample to an analysis with
a liquid chromatography tandem mass spectrometer system, said
system comprising a triple quadrupole instrument and multiple
reaction monitoring, said analysis comprising detecting an isolated
peptide of said isoform; comparing the quantitated value to a
threshold value; determining that the drug has an acceptable CYP
induction potential when the quantitated value does not exceed the
threshold value.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 12/904,520, filed Oct. 13, 2010, which claims the benefit
of priority from U.S. Provisional Patent Application Nos.
61/252,648, filed Oct. 17, 2009, and 61/252,430, filed Oct. 16,
2009, all of which are incorporated herein in their entireties by
reference.
FIELD
[0002] The present teachings relate to cytochrome P450 enzymes
(CYPs) and detection of enzymes using mass spectrometry.
BACKGROUND
[0003] Cytochrome P450 enzymes (CYPs) are major drug metabolizing
enzymes and experimental pharmaceutical compounds are generally
evaluated for their CYP induction potential early in the
development process. Measurement of a CYP induction profile in
response to a chemical can be used as a fundamental aspect of drug
safety evaluation, but expression of these proteins is regulated by
transcriptional, post transcriptional and translational mechanisms.
As a result, mRNA-based assays are not reliable predictors of CYP
induction. Further, CYPs exhibit extensive amino acid sequence
homology, particularly within subfamilies, so that P450 protein
expression methods are also poorly discriminatory.
SUMMARY
[0004] According to various embodiments of the present teachings, a
method of quantitating the 1A2, 2B6, 3A4, and 3A5 isoforms of
cytochrome P450 enzymes (CYPs) is provided without the need for any
chemical labeling. Isoform-specific tryptic peptides can be
observed in liquid chromatography-Tandem Mass Spectrometry
(LC-MSMS) analysis of samples derived from hepatocytes, for
example, in microsomes, along with their optimal Q1 and Q3
transitions. Those observed peptides and transitions, can be used
to enable a reliable CYP quantitation of the isoforms 1A2, 2B6,
3A4, and 3A5.
[0005] According to various embodiments, a set of peptides and
optimal MRM transistors are provided as "house keeping" microsomol
proteins whose concentrations are unaffected by drug incubation.
The set can be used as normalization proteins for quantitative
analysis. In some embodiments, quantitation can be performed by
spiking into the sample heavy forms of the isoform-specific
peptides, for example, forms that have been enriched with C13
and/or N15. FIG. 1 shows a typical MRM analysis for CYP 1A2,
CYP2B6, and CYP3A3/3A4 from human microsomes, wherein some of the
isoform-specific peptides have been labeled in FIG. 1. The
optimized transitions for each of a set of observed, most sensitive
isoform-specific peptides, according to various embodiments of the
present teachings, is shown in Tables 1-4 below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] The present teachings will be described with reference to
the accompanying drawings. The drawings are intended to illustrate,
not limit, the present teachings.
[0007] FIG. 1 shows an MRM analysis of 20 .mu.g of a microsomal
preparation for CYP 1A2, CYP 2B6, and CYP 3A4 (extracted ion
chromatograms (XIC) of +MRM (412 pairs)).
[0008] FIG. 2 shows an MRM analysis of 1 .mu.l of a microsomal
preparation for CYP 2B6 (extracted ion chromatogram of +MRM (15
pairs)).
[0009] FIG. 3 shows an MRM analysis of 2 .mu.g of a microsomal
preparation for CYP 1A2 (extracted ion chromatogram of +MRM (57
pairs)).
[0010] FIG. 4 shows an MRM analysis of 1 .mu.l of a microsomal
preparation for CYP 3A4 (extracted ion chromatogram of +MRM (34
pairs)).
[0011] FIG. 5A shows an MRM analysis for 30 .mu.l of a microsomal
preparation for 3A5 (extracted ion chromatogram of +MRM (48
pairs)).
[0012] FIG. 5B shows an MRM analysis for 30 .mu.l of a microsomal
preparation for 3A5 (extracted ion chromatogram of +MRM (48
pairs)).
[0013] FIG. 5C shows an MRM analysis for 30 .mu.l of a microsomal
preparation for A5 (extracted ion chromatogram of +MRM (48
pairs)).
[0014] FIG. 6 shows a graph showing protein expression changes
observed in a hepatocyte sample preparation treated with inducers
for CYP 2B6, using RNA assays, enzyme activity assays and the
protein quantitation method of the present teachings.
[0015] FIG. 7 shows a graph showing protein expression changes
observed in a hepatocyte sample preparation treated with inducers
far CYP 1A2, using RNA assays, enzyme activity assays and the
protein quantitation method of the present teachings.
[0016] FIG. 8 shows a graph showing protein expression changes
observed in a hepatocyte sample preparation treated with inducers
far CYP 3A4, using RNA assays, enzyme activity essays and the
protein quantitation method of the present teachings.
DETAILED DESCRIPTION
[0017] According to various embodiments, a method for screening a
drug for cytochrome P450 (CYP) induction is provided that comprises
incubating the drug with a microsome-containing biological sample
and quantitating at least one CYP isoform. In some embodiments, the
isoforms con comprise one or more isoform selected front 2B6, 3A4,
1A2, and 3A5 isoforms. The method can comprise using a liquid
chromatography tandem mass spectrometry (LC-MSMS) technique to
quantitate the amount of each isoform. The quantitated value of
each can be compared to a threshold value, and the drug can be
identified as having an acceptable CYP induction potential when the
quantitated value does not exceed the threshold value. The
threshold value can be selected or predetermined based on a desired
CYP induction potential or based on the CYP induction potential of
one or more different drugs, similar or non-similar to the drug
being screened. In some embodiments, the microsome-containing
biological sample can be derived from a mammal, from a primate, or
from a human.
[0018] According to various embodiments, the drug can be incubated
with a sample containing human hepatocytes. In some embodiments,
after incubation with the drag, the sample containing human
hepatocytes can be used to obtain at least one microsome fraction
by, for example, 16 G centrifugation. The microsome fraction can be
analyzed for CYP induction by the drug, by detecting isolated
peptides specific to CYP (isoform-specific peptides). According to
some embodiments, after incubation with the drug, the sample
containing human hepatocytes can be used to obtain at least one S9
fraction by, for example, 9 G centrifugation. The S9 fraction can
be analyzed to detect CYP induction by the drug, for example, by
detecting isolated peptides specific to CYP (isoform-specific
peptides). According to some embodiments, the microsome fraction or
the S9 faction can be analyzed using a liquid chromatography tandem
mass spectrometry (LC-MSMS) technique in order to quantitate at
least one CYP isoform. The quantitated value of each can be
compared to a threshold value, and the drug can be identified as
having an acceptable CYP induction potential when the quantitated
value does not exceed the threshold value.
[0019] According to various embodiments, a method is provided for
directly analyzing CYP from hepatocytes. In some embodiments,
antibody peptides can be used to pull the isoform-specific peptides
directly out of hepatocyctes. According to some embodiments, using
antibody peptides to pull the isoform-specific peptides directly
out of hepatocytes would have the advantage of not needing to
prepare S9 or microsome fractions, and would require less
hepatocyte cells for drug incubation.
[0020] In some embodiments, the method comprises comparing detected
induction to a control. For example, became little or no drug
induction of CYPs is desirable, a threshold can be set such that
the drug mug show less than (<) 40% induction compared to the
positive control, to be considered acceptable. In some embodiments,
the drug must show less than (<) 30% induction compared to the
positive control, to be considered acceptable. In other
embodiments, the drug mast show less than (<) 20% induction
compared to the positive control, to be considered acceptable.
[0021] According to various embodiments, a method for determining
an amount of at least one isoform of cytochrome P450 (CYP) in a
sample, is provided. The method can comprise the use of a mass
spectrometry technique, wherein the at least one isoform of
cytochrome P450 comprises at least one of CYP 2B6, CYP 3A4, CYP
1A2, and CYP 3A5. The mass spectrometry technique can comprise a
tandem mass spectrometry (MS/MS) technique and/or a liquid
chromatography tandem mass spectrometry (LC-MS/MS) technique. In
some embodiments, the technique comprises an LC-MS/MS technique and
the use of a triple quadrupole instrument and Multiple Reaction
Monitoring (MRM).
[0022] FIG. 1 shows a typical MRM analysis for CYP 1A2, CYP 2B6,
and CYP 3A4, well as the "housekeeping" Microsomal proteins
(Microsomal GST, Corticosteroid 11 beta, and Microsomal
Tryglyceride), from a microsomal sample preparation prepared as
described below in the Examples. The quantity of CYP 1A2, CYP 2B6,
and CYP 3A4 can be determined by, for example, Isotope Dilution
Mass Spectrometry, wherein the sample preparation is spiked with
heavy forms of the isoform-specific peptides. The quantity of CYP
1A2, CYP 2B6, and CYP 3A4 can also be determined using other
conventional methods known in the art. In some embodiments, the
method uses LC-MSMS with multiple reaction monitoring (MRM)
quantitation of the isoform-specific peptides and isotope-coded
affinity tags (ICAT) to generate a CYP induction profile. The
method can use, for example, approaches similar to the approaches
presented by Pennington et al. to quantitate isoform-specific
cysteine-containing peptides labeled with ICAT as described in
Proteomics, 6(6), pages 1934-1947 (March 2006), which is
incorporated herein in its entirety by reference.
[0023] FIG. 2 shows a typical MRM analysis for CYP 2B6 from the
microsomal preparation prepared a described herein. Isolated
peptides comprising the amino acid sequence of SEQ ID NOS: 1, 2, 3,
or 4 identified in Table 1 below are specific to CYP 2B6.
[0024] FIG. 3 shows a typical MRM analysis for CYP 1A2 from the
microsomal preparation prepared as described herein. The isolated
peptides comprising the amino acid sequence of SEQ ID NOS: 8, 9,
10, 11, 12, 13, or 14 identified in Table 3 below are specific CYP
1A2.
[0025] FIG. 4 shows a typical MRM analysis for CYP 3A4 from the
microsomal preparation prepared as described herein. The isolated
peptides comprising the amino acid sequence of SEQ ID NOs: 5, 6, or
7 identified in Table 2 below are specific to CYP 3A4. It should be
understood that peptides comprising the amino acid sequence of SEQ
ID NOS: 5, 6, or 7 can also be used to identify and/or quantify CYP
3A3.
[0026] The isolated peptides comprising the amino acid sequence of
SEQ ID NOs: 15, 15, or 17 identified in Table 4 below are specific
to CYP 3A5.
[0027] FIGS. 5A-5C show three different panes of a typical MRM
analysis for CYP 3A5 from the microsomal preparation prepared as
described herein. Each pane relates to specific transitions used
for the particular peptide. The isolated peptides comprising the
amino acid sequence or SEQ ID NOS: 15, 16 or 17 are specific to CYP
3A5.
[0028] According to various embodiments, the method can comprise
determining an amount or CYP 2B6 in the sample by detecting an
isolated peptide specific to cytochrome P450 (CYP) isoform CYP 2B6,
for example, one or more of the isoforms comprising the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 identified
herein. The amount can be determined using a triple quadrupole
instrument and Multiple Reaction Monitoring (MRM). In some
embodiments, the isolated peptide can comprise the amino acid
sequence of SEQ ID NO: 1 identified herein, and the method can
comprise monitoring precursor-product ion pair transitions having
an m/z value of about 548/911, 548/681, or 548/566, wherein the
term "about" as used herein means within a range of +/- one (1)
atomic mass unit. In some embodiments, the isolated peptide can
comprise the amino acid sequence of SEQ ID NO: 2 identified herein,
and the method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 494/777, 494/437, or
494/874. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 3 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 421/508, 421/607, or
421/694. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 4, and the method can comprise
monitoring precursor-product ion pair transitions having an m/z
value of about 479/499, 479/614, or 479/727.
[0029] According to various embodiments, the method can comprise
determining an amount of CYP 3A4 in the sample by detecting an
isolated peptide specific to cytochrome P450 (CYP) isoform CYP 3A4,
comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6,
or SEQ ID NO: 7 identified herein. The method can use, for example,
a triple quadrupole instrument and Multiple Reaction Monitoring
(MRM). In some embodiments, the isolated peptide can comprise the
amine acid sequence of SEQ ID NO: 5 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 440/549, 440/650, or
440/532. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 6 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 704/794, 704/929, 564/639,
564/745, or 564/790. In some embodiments, the isolated peptide can
comprise the amino acid sequence of SEQ ID NO: 7 identified herein,
and the method can comprise monitoring precursor-product ion per
transitions having an m/z value or about 798/819, 798/932, or
798/1004.
[0030] According to various embodiments, the method can comprise
determining en amount of CYP 1A2 in the sample by detecting an
isolated peptide specific to cytochrome P450 (CYP) isobar CYP 1A2,
comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ
ID NO: 14, identified herein. The method can use, for example, a
triple quadrupole instrument and Multiple Reaction Monitoring
(MRM).
[0031] In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 8 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 432/636, 432/535, or
432/478. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 9 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 482/800, 482/628, or
482/743. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 10 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 491/721, 491/834, or
491/535. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 11 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 528/501, 528/614, or
528/727. In some embodiments, the isolated peptide can comprise the
amino acid sequence of SEQ ID NO: 12 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 571/783, 571/971,
571/1028, 381/587, 381/474, or 381/375. In some embodiments, the
isolated peptide can comprise the amino acid sequence of SEQ ID NO:
13 identified herein, and the method can comprise monitoring
precursor-product ion pair transitions having an m/z value of about
695/695, 695/837, or 695/950. In some embodiments, the isolated
peptide can comprise the isolated peptide comprises the amino acid
sequence of SEQ ID NO: 14 identified herein, and the method can
comprise monitoring precursor-product ion pair transitions having
an m/z value of about 536/795, 536/584, or 536/698.
[0032] According to various embodiment the method can comprise
determining an amount of CYP 3A5 in the sample by detecting an
isolated peptide specific to cytochrome P450 (CYP) isoform CYP 3A5,
comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16,
or SEQ ID NO: 17 identified herein. The method can use, for
example, a triple quadrupole instrument and Multiple Reaction
Monitoring (MRM). In some embodiments, the isolated peptide can
comprise an amino acid sequence of SEQ ID NO: 15 identified herein,
and the method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 468/581, 468/679, or
468/736. In some embodiments, the isolated peptide can comprise on
amino acid sequence of SEQ ID NO: 16 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 470/494, 470/608, or
470/722. In some embodiments, the isolated peptide can comprise an
amino acid sequence of SEQ ID NO: 17 identified herein, and the
method can comprise monitoring precursor-product ion pair
transitions having an m/z value of about 589/747, 589/696, or
589/647.
[0033] According to various embodiments of the present teachings, a
kit is provided that can comprise one or more of the isolated
peptides specific to one or more of cytochrome P450 (CYP) isoform
CYP 2B6, CYP 3A4, CYP 1A2, and CYP 3A5. For example, the kit can
comprise one or more isolated proteins specific to CYP 2B6,
comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, or SEQ ID NO: 4. The kit can comprise one or more
isolated proteins specific to CYP isoform CYP 3A4, comprising the
amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
The kit can comprise one or more isolated proteins specific to CYP
isoform CYP 1A2, comprising the amino acid sequence of SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ
ID NO: 13, or SEQ ID NO: 14. The kit can comprise one or more
isolated proteins specific to CYP isoform CYP 3A5, comprising the
amino acid sequence of SEQ ID NO: 15, SEQ ID NO 16, or SEQ ID NO:
17.
[0034] In some embodiments, the kit can comprise at least one
isolated peptide specific to each of CYP isoforms CYP 2B6, CYP 3A4,
CYP 1A2, and CYP 3A5. For example, the kit can comprise each of the
isolated peptides of SEQ ID NOS: 1-17 identified herein, and
further can comprise instructions for measuring Q1 and Q3
transition values for each of the isoform-specific peptides. The
kit can comprise enzyme digestion components including buffers and
enzymes, other buffers, and optionally other reagents and/or
components. In some embodiments, the kit can comprise, for example,
a homogeneous assay such that the user need only add a sample. In
some embodiments, the kit can comprise calibration or normalization
reagents or standards. Information pertaining to instrument
settings that can or should be used to perform an assay can also be
included in the kit. Information pertaining to sample preparation,
operating conditions, volumetric amounts, temperature settings, and
the like, can be included with the kit.
[0035] According to various embodiments, different transitions can
be used to measure and benchmark assay results, depending on
various factors. Accordingly, the kit can comprise different
transition values and/or suggested settings, useful to make
comparative measurements between a sample and one or more control
reagents. The kit can include instructions to measure specific
pairs of transition values, for example, the Q1/Q3 transition pair,
or the values of one or more different transition pairs.
[0036] The kit can be packaged in a hermetically sealed container
containing one or more regent vessels and appropriate instructions.
An electronic medium can be included in the kit, having stored
thereon electronic information pertaining to one or more assays,
measurement values, transition pairs, operating instructions,
software for carrying out operations, a combination thereof, or the
like.
EXAMPLES
[0037] The present teachings can be even more fully understood with
reference to the examples and resulting data that follow. In the
examples below and the results shown in the attached drawing
figures, a CYP induction study was undertaken as follows.
Hepatocyte Treatment
[0038] Primary cultures of humus hepatocytes were treated for 72
hours with the prototypical liver enzyme inducers
3-methylcholanthrene (3-MC, 2 .mu.M), phenobarbital (PB, 1 mM), or
rifampicin (RIF, 10 .mu.M) to enhance the expression and activity
of CYP1A2, CYP2B6, and CYP3A4, respectively. The CYP quantitation
method of the present teachings was used to determine the amount of
CYP in the sample after treatment with the liver enzyme inducers.
In addition, CYP activity was measured by metabolite formation from
selective substrates (phenacetin, bupropion and testosterone,
respectively) and mRNA was measured by qRT-PCR (Taqman.RTM.,
Applied Biosystems).
Microsome Preparation
[0039] Microsomal subcellular fractions were prepared by lysing
treated hepatocytes in homogenization buffer (50 mM TRIS-HCl, pH
7.0, 150 mM KCl, 2 mM EDTA) followed by centrifugation at
9,000.times.g for 20 minutes at 4.degree. C. The supernatant (S9
fraction) was then spun at 100,000.times.g for 60 minutes at
4.degree. C. The resulting microsomal pellet was resuspended in
0.25 M sucrose and stored at -80.degree. C. until analysis.
Tryptic Digestion
[0040] To 100 ul of each microsomal preparation, 5 ul 2% SDS was
added, followed by 10 ul of 50 mM TCEP and incubated at 60.degree.
C. for 1 hr, 5 ul of 0.1M MMTS was added and incubated at room
temperature for 10 minutes. 100 ul of 100 mM TRIS (pH=8.50) was
then added followed by 50 ug of trypsin and the resultant solution
was digested overnight at 37.degree. C. On (1) ng of each
isotopically enriched synthetic peptide was added to the digest and
analyzed by LC-MS.
Chromatography
[0041] Chromatography was performed using an Agilent 1100 system
(Agilent of Santa Clara, Calif.) coupled to a C18 Jupiter Proteo
50.times.2.0 mm column (Phenomenex of Torrance, Calif.). The
gradient was 5-40% B over 15 minutes with A consisting of 2% ACN,
0.1% formic and B consisting of 90% ACN, 10% H2O, 0.1% formic acid.
Flow rate was 700 .mu.L/min.
Mass Spectrometry
[0042] Samples were analyzed on a Applied Biosystems MDS SCIEX 4000
QTRAP.TM. LC/MS/MS system, using a Turbo V.TM. source and Analyst
1.5. For quantitation, scheduled MRM (sMRM) was used to maximize
dwelt time on each transition.
Data Processing
[0043] Quantitative data was processed using MultiQuart.TM. 1.2
software available from Applied Biosystems, LLC of Foster City,
Calif.
Results
[0044] FIG. 6 is a graph comparing the changes in expression of CYP
2B6 observed using the RNA assay, the CYP activity assay
(designated "enzyme activity assay" in the figures), and a CYP
quantitation method of the present teachings (designated "protein
assay" in the figures). The 3-MC is a vehicle control and induces
basal levels of CYP 2B6. The PB is a prototypical inducer for CYP
2B6, by CAR nuclear inceptor activation. The RIF is also a known
inducer of CYP 2B6. As is shown, protein expression changes
observed in the CYP activity assay and RNA assay generally mirror
the expression changes observed using a CYP quantitation method of
the present teachings.
[0045] FIG. 7 is a graph comparing the changes in expression of CYP
1A2 observed using the RNA assay, the CYP activity assay
(designated "enzyme activity assay" in the figures), and the CYP
quantitation method of the present teachings (designated "protein
assay" in the figures). The 3-MC is a prototypical inducer of CYP
1A2 by AhR nuclear receptor activation. The PB and RIP are vehicle
controls inducing basal levels of 1A2, if any. As is shown, protein
expression changes observed in the CYP activity assay and RNA assay
generally mirror the expression changes observed using a CYP
quantitation method of the present teachings, except that the RNA
way for the sample treated with 3MC exhibits very high levels of
RNA. The RNA assay cannot accurately quantify proteins because not
all mRNA is converted to protein.
[0046] FIG. 8 is a graph comparing the changes in expression of CYP
3A4 observed using the RNA assay, the CYP activity assay
(designated "enzyme activity assay" in the figures), and a CYP
quantitation method of the present teachings (designated "protein
assay" in the figures). The 3-NIC minimally induces CYP 3A4. The PB
significantly inducts 3A4. The RIF is a prototypical inducer of CYP
3A4 by PXR nuclear receptor activation. As is shown, protein
expression changes observed in the CYP activity any and RNA assay
generally mirror the expression changes observed using the CYP
quantitation method of the present teachings.
[0047] Table 1 below shows sequences of the peptides determined,
according to the present teachings, to be specific to cytochrome
P450 (CYP) isoform CYP 2B6, along with their optimal MRM Q1, Q3
transitions. According to various embodiments, these observed
peptides and transitions can be used to enable a reliable CYP
quantitation of the isoform CYP 2B6.
TABLE-US-00001 TABLE 1 2B6 Human O1 O3 IAMVDPFFR 548.3 911.4 (SEQ
ID NO: 1) 548.3 681.3 548.3 566.3 IPPTYQIR 494.3 777.4 (SEQ ID NO:
2) 494.3 437.7 494.3 874.5 FSVTTMR 421.2 508.4 (SEQ ID NO: 3) 421.2
607.3 421.2 694.4 ETLDPSAPK 479.2 499.3 (SEQ ID NO: 4) 479.2 614.3
479.2 727.4
[0048] Table 2 below shows sequences of the peptides determined,
according to the present teachings, to be specific to cytochrome
P450 (CYP) isoform CYP 3A4, along with their optimal MRM Q1, Q3
transitions. According to various embodiments, these observed
peptides and transitions can be used to enable a reliable CYP
quantitation of the isoform CYP 3A4.
TABLE-US-00002 TABLE 2 3A4 Human O1 O3 EVTNFLR 439.7 549.3 (SEQ ID
NO: 5) 439.7 650.5 439.7 532.3 LSLGGLLQPEKPVVLK 704.4 794.5 (SEQ ID
NO: 6) 704.4 929.8 LSLGGLLQPEKPVVLK +3 564.3 789.5 (SEQ ID NO: 6)
564.3 745.9 564.3 689.4 VWGFYDGQQPVLAITDPDMIK 798.4 819.4 (SEQ ID
NO: 7) 798.4 932.5 798.4 1003.5
[0049] Table 3 below snows sequences of the peptides determined,
according to the present teachings, to be specific to cytochrome
P450 (CYP) isoform CYP 1A2, along with their optimal MRM Q1, Q3
transitions. According to various embodiments, these observed
peptides and transitions can be used to enable a reliable CYP
quantitation of the isoform CYP 1A2.
TABLE-US-00003 TABLE 3 1A2 Human O1 O3 DITGALFK 432.7 636.4 (SEQ ID
NO: 8) 432.7 535.3 432.7 478.3 YGDVLQIR 482.3 800.5 (SEQ ID NO: 9)
482.3 628.4 482.3 743.4 FLWFLQK 491.3 721.4 (SEQ ID NO: 10) 491.3
834.4 491.3 535.3 ASGNLIPQEK 528.7 501.2 (SEQ ID NO: 11) 528.7
614.4 528.7 727.4 IGSTPVLVLSR +2 571.4 783.5 (SEQ ID NO: 12) 571.4
971.6 571.4 1028.6 IGSTPVLVLSR +3 381.1 587.4 (SEQ ID NO: 12) 381.1
474.3 381.1 375.2 SPPEPWGWPLLGHVLYGK 695.4 695.9 (SEQ ID NO: 13)
695.4 837.5 695.4 950.5 YLPNPALQR 536.3 795.4 (SEQ ID NO: 14) 536.3
584.3 536.3 698.4
[0050] Table 4 below shows sequences of the peptides determined,
according to the present teachings, to be specific to cytochrome
P450 (CYP) isoform CYP 3A5, along with their optimal MRM Q1, Q3
transitions. According to various embodiments, these observed
peptides and transitions can be used to enable a reliable CYP
quantitation of the isoform CYP 3A5.
TABLE-US-00004 TABLE 4 3A5 O1 O3 SLGPVGFMK 468.5 581.5 (SEQ ID NO:
15) 468.5 678.1 468.5 735.5 DTINFLSK 469.5 494.3 (SEQ ID NO: 16)
469.5 608.4 469.5 721.5 GSMVVIPTYALHHDPK 589.2 746.5 (SEQ ID NO:
17) 589.2 696 589.2 646.5
[0051] Table 5 below shows sequences of the peptides along with
their optimal MRM Q1, Q3 transitions for the house-keeping
microsomal protein Microsomal GST. According to various
embodiments, the concentration of this observed peptide is
unaffected by drug incubation and thus the peptide can be useful as
a normalization protein to enable reliable CYP quantitation.
TABLE-US-00005 TABLE 5 Microsomal GST O1 O3 DVNVENVNQQR 657.8 758.3
(SEQ ID NO: 18) 657.8 887.5 657.8 545.3 MYLLALK 426.3 720.5 (SEQ ID
NO: 19) 426.3 557.4 426.3 444.3 NALLPEGIPSLLK 682.9 1066.7 (SEQ ID
NO: 20) 682.9 727.5 682.9 953.6
[0052] Table 6 below shows sequences of the peptides along with
their optimal MRM Q1, Q3 transitions for the house-keeping
microsomal protein Microsomal Triglyceride. According to various
embodiments, the concentration of this observed peptide is
unaffected by drug incubation and thus the peptide can be useful as
a normalization protein to enable reliable CYP quantitation.
TABLE-US-00006 TABLE 6 Microsomal Tryglyceride O1 O3 MMLMSTATAFYR
711.8 829.4 (SEQ ID NO: 21) 711.8 916.2 711.8 1047.5
[0053] Table 7 below shows sequences of the peptides along with
their optimal MRM Q1, Q3 transitions for the house-keeping
microsomal protein Corticosteroid 11 beta. It should be understood
that the Q1 and Q3 masses for the peptide of SEQ ID NO: 22 in Table
7 refers to the MMTS alkylated peptide and that changing to a
different alkylating reagent will change the Q1, Q3 masses. In
addition, alkylating reagents other than MMTS can be used.
According to various embodiments, the concentration of this
observed peptide is unaffected by drug incubation and thus the
peptide can be useful as a normalization protein to enable reliable
CYP quantitation.
TABLE-US-00007 TABLE 7 Corticosteroid 11 beta O1 O3 EECALEIIK 547.3
615.4 (SEQ ID NO: 22) 547.3 686.4 547.3 835.4 FALDGFFSSIR 630.3
928.4 (SEQ ID NO: 23) 630.3 1041.5 630.3 813.4
[0054] The observed isoform-specific tryptic peptides that are
detected using LC-MSMS analysis of microsomes, along with their
optimal Q1, Q3 transitions, enable a method for CYP quantitation of
the isoforms 1A2, 2B6, 3A4, and 3A5 without the need for any
chemical labeling approaches.
[0055] Other embodiments of the present teachings will be apparent
to the skilled in the art from consideration of the present
specification and practice of the present teachings disclosed
herein. It is intended that the present specification and examples
be considered as exemplary only.
Sequence CWU 1
1
2319PRTHuman 1Ile Ala Met Val Asp Pro Phe Phe Arg 1 5 28PRTHuman
2Ile Pro Pro Thr Tyr Gln Ile Arg 1 5 37PRTHuman 3Phe Ser Val Thr
Thr Met Arg 1 5 49PRTHuman 4Glu Thr Leu Asp Pro Ser Ala Pro Lys 1 5
57PRTHuman 5Glu Val Thr Asn Phe Leu Arg 1 5 616PRTHuman 6Leu Ser
Leu Gly Gly Leu Leu Gln Pro Glu Lys Pro Val Val Leu Lys 1 5 10 15
721PRTHuman 7Val Trp Gly Phe Tyr Asp Gly Gln Gln Pro Val Leu Ala
Ile Thr Asp 1 5 10 15 Pro Asp Met Ile Lys 20 88PRTHuman 8Asp Ile
Thr Gly Ala Leu Phe Lys 1 5 98PRTHuman 9Tyr Gly Asp Val Leu Gln Ile
Arg 1 5 107PRTHuman 10Phe Leu Trp Phe Leu Gln Lys 1 5 1110PRTHuman
11Ala Ser Gly Asn Leu Ile Pro Gln Glu Lys 1 5 10 1211PRTHuman 12Ile
Gly Ser Thr Pro Val Leu Val Leu Ser Arg 1 5 10 1319PRTHuman 13Ser
Pro Pro Glu Pro Trp Gly Trp Pro Leu Leu Gly His Val Leu Thr 1 5 10
15 Leu Gly Lys 149PRTHuman 14Tyr Leu Pro Asn Pro Ala Leu Gln Arg 1
5 159PRTHuman 15Ser Leu Gly Pro Val Gly Phe Met Lys 1 5 168PRTHuman
16Asp Thr Ile Asn Phe Leu Ser Lys 1 5 1716PRTHuman 17Gly Ser Met
Val Val Ile Pro Thr Tyr Ala Leu His His Asp Pro Lys 1 5 10 15
1811PRTHuman 18Asp Val Asn Val Glu Asn Val Asn Gln Gln Arg 1 5 10
197PRTHuman 19Met Tyr Leu Leu Ala Leu Lys 1 5 2013PRTHuman 20Asn
Ala Leu Leu Pro Glu Gly Ile Pro Ser Leu Leu Lys 1 5 10 2112PRTHuman
21Met Met Leu Met Ser Thr Ala Thr Ala Phe Tyr Arg 1 5 10
229PRTHuman 22Glu Glu Cys Ala Leu Glu Ile Ile Lys 1 5 2311PRTHuman
23Phe Ala Leu Asp Gly Phe Phe Ser Ser Ile Arg 1 5 10
* * * * *