RESUMO
The final goal of the Russian part of the Chromosome-centric Human Proteome Project (C-HPP) was established as the analysis of the chromosome 18 (Chr 18) protein complement in plasma, liver tissue and HepG2 cells with the sensitivity of 10(-18) M. Using SRM, we have recently targeted 277 Chr 18 proteins in plasma, liver, and HepG2 cells. On the basis of the results of the survey, the SRM assays were drafted for 250 proteins: 41 proteins were found only in the liver tissue, 82 proteins were specifically detected in depleted plasma, and 127 proteins were mapped in both samples. The targeted analysis of HepG2 cells was carried out for 49 proteins; 41 of them were successfully registered using ordinary SRM and 5 additional proteins were registered using a combination of irreversible binding of proteins on CN-Br Sepharose 4B with SRM. Transcriptome profiling of HepG2 cells performed by RNAseq and RT-PCR has shown a significant correlation (r = 0.78) for 42 gene transcripts. A pilot affinity-based interactome analysis was performed for cytochrome b5 using analytical and preparative optical biosensor fishing followed by MS analysis of the fished proteins. All of the data on the proteome complement of the Chr 18 have been integrated into our gene-centric knowledgebase ( www.kb18.ru ).
Assuntos
Cromossomos Humanos Par 18 , Bases de Dados de Proteínas , Proteoma/análise , Proteínas Sanguíneas/classificação , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 18/metabolismo , Expressão Gênica , Genoma Humano , Células Hep G2 , Humanos , Fígado/metabolismo , Espectrometria de Massas , TranscriptomaRESUMO
Sequential thin slicing of one-dimensional electrophoresis gels followed by slice-by-slice mass spectrometry to allow protein identification was used to produce a proteomic map for cytochromes P450. Parallel MALDI-TOF-MS and LC-MS/MS analyses were performed. Combination of the two MS methods increased the quality of protein identification. We have proposed an efficient approach to obtain a comprehensive profile of drug-metabolizing enzymes in the liver that can be used to differentiate between polymorphic variants of cytochromes P450.
