The Insight into the Human Liver Proteome

Fuchu He   Consortium of Chinese Human Liver Proteome Project;  State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China; Institutes of Biomedical Sciences, Fudan University, Shanghai, China

Overview

The Human Liver Proteome Project (HLPP) is one of the large-scale international collaborative initiatives focusing on the proteomic analysis of the human liver which was launched by the Human Proteome Organization (HUPO). It aims to generate a comprehensive protein atlas of the human liver, uncover the proteomic basis of liver development, physiology and pathology and develop the liver-specific diagnostics and therapeutics.

On the basis of proposals from participant scientists and extensive discussion, the scientific categories and objectives were finally defined as eight subprojects: Sampling and Banking, Expression Profiling, Protein Linkage (Protein-Protein Interaction) Mapping and ORFeome, Modification Profiling, Localization Mapping, Antibody Banking, Bioinformatics, and Liver Diseases.

In the past five years, this project has set up standard operating procedures and optimized the strategies of the proteomic analysis. In order to globally catalog the proteome in the human liver, an effective strategy was utilized to deal with its complexity. First of all, the fresh samples from the adult liver were carefully collected in strict accordance with the ethical rules of tissue collection and the standard operation procedures (SOPs). Secondly, due to the high contents of a proteome and lack of a panacea for its analysis, an integrated platform was established. Finally, a powerful bioinformatics platform was constructed to deal with the flooding proteomic data. In the light of these strategies, we identified 2 495 unique proteins in the Chinese fetal liver tissues, 6 436 in the French adult liver tissues and 12 951 in Chinese adult liver tissues respectively.

Recently, we have turned to the proteomic analysis of the different organelles as well as the different kinds of liver cells. We succeeded in identifying 5882 proteins with 2 peptides or more in 95% confidence from plasma membrane, nucleus, cytoplasm, mitochondria, rough endoplasmic reticulum and smooth endoplasmic reticulum in human liver cells, which will be compared with those proteins identified from the human liver tissues. In addition, 4969 proteins were identified from the same organelles of the mouse liver. Furthermore, we optimized the approach of the extracorporeal liver perfusion and cell sorting and obtained the purified mouse hepatic parenchymal cells, in which, 2216 proteins were identified with at least 2 peptides in 99% confidence.

In the meantime, other subprojects of the HLPP have also made lots of progress, such as the ORFeome, protein-protein interactions, antibodies, post-translational modifications, localizations, and some proteomic analysis of the human liver diseases. 5500 ORFs of genes expressed in human liver have been collected in an ORF bank. 1732 unique protein-protein interactions have been found by screening the human liver cDNA library with the yeast two-hybrid method. Meanwhile, a protein array of about 5000 unique liver ORFs was screened by yeast two hybrid mating method and 1632 protein-protein interactions were discovered. Of  them, 1270 interactions were verified by yeast retransformation assay. More than 2000 hybridoma cell lines were established and classified, in which 1500 or so were characterized as mAbs for more than 200 different proteins. The proteomic changes in liver tissues and plasma were studied and compared during the progression of the liver diseases, from the “normal liver” to hepatitis, hepatic fibrosis, hepatocirrhosis, HCC staging and its metastasis. Tens of potential biomarkers for liver disease have been found and are being further validated. Other sub-projects, such as the post-translational modifications and proteome localizations were focused on the technology development and also progressed steadily.

As a promising perspective, it will establish the “Solar System” of Human Liver Proteome by the end of the action phase and furthermore make great progress in diagnostics and therapeutics of the liver diseases in the future.