Characterisation of the liver and serum proteomes is the next step in the investigation of patients with liver diseases since protein signatures are a potentially powerful tool in the diagnosis and prognosis of patients with HCV infection. Although there are several studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been extensively characterised. Proteomics uses a combination of sophisticated techniques including 2D gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing and bioinformatics.78
A major hurdle when studying proteins is the potentially enormous number of proteins present in a biological sample. This diversity is due to the many post-transcriptional and post-translational modifications that proteins can display. Another difficulty is the wide range of concentrations of highly represented proteins and the low concentration of potential biomarkers. Without specific protein amplification techniques such as PCR for nucleic acids, the identification of biomarkers requires technologies which have not yet been fully validated. However, there are several good reasons to focus on protein analysis: (1) the level of mRNA expression does not often parallel the amount of protein; (2) study of the genome does not address dynamic cell processes; and (3) recent improvements in proteomic technologies, such as proteomic profiling technology, provide global visualisation of the proteome by a high-throughput method and lead to the identification of isolated or clustered peaks associated with a disease in a complex biological sample.
There are very few studies in the literature addressing proteomic changes in hepatitis C. Jacobs et al
provide a global proteome analysis of changes induced by HCV infection by multidimensional liquid chromatographic separation coupled with mass spectrometry in the full-length HCV replicon model.79
Several proteins involved in lipid biosynthesis were found to be upregulated, while proteins involved in fatty acid oxidation were downregulated. Some of these proteins were also found to be deregulated in liver biopsies of patients with chronic HCV infection. These data support the suggested relationship between HCV and lipid metabolism, although the physiopathological relevance of these data needs to be investigated further.
Liver fibrosis and cirrhosis are the consequences of chronic HCV infection. They are characterised by marked modifications of proteins including synthesis of extracellular matrix proteins, suggesting that proteomics might provide new insights for diagnosis of fibrosis or cirrhosis. Several studies have been performed to characterise protein changes in tissues in experimental models of liver fibrosis. For instance, several proteins that are significantly deregulated during liver fibrosis have been identified by 2D gel electrophoresis and mass spectrometry.80
Using similar experimental animal models and culture of hepatic stellate cells (HSCs), Kristensen et al
compared proteomic dynamics in in vitro and in vivo processes of HSC activation.81
The expression of 43 proteins was shown to be altered by 2D gel electrophoresis, when the cells were activated in vivo and/or in vitro. Among them, 27 displayed similar changes in vivo and in vitro, including two members of the S100 protein family (calcyclin and calgizzarin) and galectin-1 (a galactosidase-binding lectin) involved in growth regulation and neoplastic transformation.
To date, few studies have focused on serum protein changes during the development of liver fibrosis. In thioacetamide-induced liver fibrosis, Xu et al
showed that expression of 59 protein spots significantly changed upon thioacetamide administration and that a protein of 3.495 kDa, sharing homology with a histidine-rich glycoprotein, was consistently decreased in sera of cirrhotic rats.82
In addition, in one study, Poon et al
tried to define serum protein signatures associated with various degrees of fibrosis and develop a proteomic fingerprinting model for predicting fibrosis and cirrhosis in patients with chronic hepatitis B infection.83
For this purpose, they developped ANN models to generate an ANN fibrosis index based on the proteomic data obtained by ProteinChip Array with laser desorption-ionisation time of flight mass spectrometry (SELDI-TOF-MS ProteinChip technology). SELDI-TOF provides rapid protein profiles and comparative analyses according to patient phenotype.83
This approach takes advantage of an initial fractionation step of the proteome according to fixation on different affinity surfaces, followed by desorption and time-of-flight analysis of retained proteins. This technique has several advantages such as being easy to use and high throughput, making it compatible with clinical proteomics. Thirty protein changes were defined that were significantly associated with the extent of liver fibrosis.83
Interestingly, some of these protein peaks were correlated with biological data exploring liver function, such as albumin. Finally, this study showed that the ANN fibrosis index derived from the serum proteomic fingerprint predicts patients with significant fibrosis and cirrhosis. Using the same approach, we demonstrated that antiviral treatment induced chronological changes in the serum proteome and that these variations were dependent upon virological response to treatment.84
Moreover, serum proteome analysis in naive patients predicted SVR to PEG-IFN plus ribavirin in most of our patients. Molecular identification of the peaks isolated by the SELDI-TOF approach would be of major interest for developing serum tests for patient care. Although these global approaches are quite effective and are well adapted to clinical proteomics. they have several limitations including the difficult molecular identification of protein peaks and the necessity for further analysis based on purification, peptide fingerprinting and MS/MS sequencing. This is the major limitation of global proteomics analyses at present.