Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content.

[Analysis of protein phosphorylation by combination of IMAC, phosphatase with biological mass spectrometry].

Protein phosphorylation is the most important reversible post-translational modification in cells. Analysis of phosphorylated proteins and identification of their phosphorylation sites is helpful for understanding their biological functions. MALDI-TOF-MS and ESI-Q-TOF-MS play important roles in protein phosphorylation analysis. In this work, immobilized metal affinity chromatography (IMAC) was used to selectively enrich phosphopeptides from protein digest mixtures, and treatment of phosphopeptides with alkaline phosphatase was used to confirm the phosphorylation. Finally, the phosphorylation sites were determined by tandem mass spectrometry analysis and database searching.

[Comparison of the separation performances between semi-preparative chromatographic column and chromatographic pie].

By comparing the resolution and recoveries of proteins between semi-preparative chromatographic column and chromatographic pie (pie-like column) having the same geometric volume under the same chromatographic conditions, the result from the former was found to have the almost same as that form the latter. However, the back pressure of the chromatographic pie rose slower than that of the chromatographic column with the increase of flow rate. Furthermore, the mass and volume loads for the chromatographic pie were higher than those for the chromatographic column. The results indicated that the chromatographic pie had more advantages than the chromatographic column in their performances, and this lay a foundation of the applications of the chromatographic pie in industry.