Quantitative analysis of cytochrome P450 isozymes by means of unique isozyme-specific tryptic peptides: A proteomic approach
Quantitative analysis of cytochrome P450 isozymes by means of unique isozyme-specific tryptic peptides: A proteomic approach
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DOI：
10.1124/dmd.105.004812
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发表时间：
2005-09-01
影响因子：
3.9
通讯作者：
Yakovlev, D
中科院分区：
文献类型：
--
作者：
Alterman, MA;Kornilayev, B;Yakovlev, D
建立了一种新的基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱学方法来定量细胞色素P450(P450)同工酶。结果表明，无论是在简单的双肽混合物中，还是在复杂的消化混合物中，P450同工酶特异性多肽的摩尔比都与相应多肽的质量峰面积比成线性关系。这种方法既适用于凝胶内消化(如CYP2B1和CYP2B2所示)，也适用于溶液中消化(如CYP1A2、CYP2E1和CYP2C19所示)，并且不需要引入稳定的同位素或用同位素编码的亲和标记进行标记。用合成的P450同工酶特异性多肽标准品绘制相应的校正曲线，即可进行相对和绝对定量。以CYP2B2同工酶特异性多肽(1306.7 Da)为通用内标，进行人P450同工酶的绝对定量。对两个高度同源(>；97%)的大鼠肝脏细胞色素P450同工酶和三个人类P450同工酶进行了验证，它们分别属于两个不同的家族和三个不同的亚家族：细胞色素P1A2、细胞色素P2E1和细胞色素P2C19。综上所述，我们已经证明了基于MALDI TOF的不同细胞色素P450同工酶的多肽质量指纹图谱不仅可以提供定性数据，还可以提供定量数据。
A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.