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.

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.