Disease proteomics

2008 Genomic Medicine  
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is frequently associated with cirrhosis, chronic hepatitis B (HBV) and C (HCV) virus infection. The objective of our study was to identify altered protein expression or glycosylation for use as biomarkers in HCC. Quantitation of proteins in complex biological samples using mass spectrometry has become feasible in recent years using methods that rely on incorporation of stable isotopes into peptides. Our approach was to
more » ... use multiple technologies including the multiplexed isotope labeling for relative quantitation of HCC associated proteins using iTRAQ method and 18 O based quantitation and identification of tumor specific glycosylated proteins for the discovery phase and Western blotting and immunohistochemical labeling of HCC tissues for the validation phase. Two dimensional LC-MS/MS analysis and glycoprotein enrichment strategy generated more than 50,000 MS/MS spectra from which we were able to identify and quantitate more than 1,000 liver proteins in hepatocelluar carcinoma. Over 200 proteins were found to be upregulated in HCC, which included both previously described markers as well as novel ones. Novel overexpressed proteins in HCC included fibroleukin, myeloidassociated differentiation marker, prothymosin alpha, high mobility group AT-hook 1 isoform a and leucine rich repeat containing 59. Over 100 proteins were found to be downregulated in HCC relative to adjacent normal tissues which included urea cycle enzymes, class I and class II alcohol dehydrogenases, fatty acid binding protein 1 and prostatic binding proteins. Western blotting confirmed the results of both upregulated and downregulated proteins. Using immunohistochemical labeling, we were able to validate overexpression of fibroleukin, myeloid-associated differentiation marker and fetuin in HCC and downregulation of urea cycle enzymes, fatty acid binding protein and prostatic binding proteins in HCC using tissue microarrays (n = 60). Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. Using lectin affinity followed by PNGase F digestion coupled to 18 O labeling, we identified 34 glycosylation sites. This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC. Stem cells have great potential for developing new approaches for the treatment of degenerative diseases including neurons, skeletal and cardiac muscles, beta cells of pancreas, and cancer cells. Cell replacement therapy with stem cell and their derivatives are thus an important biomedical tool. A better understanding of the molecular pathways, regulatory networks and their dynamics, which determine their diverse differentiation fates, is needed for these therapeutic approaches to be successful. With these objectives, we have been studying protein expression in mouse embryonic stem cell lines (R1-9 and ABI), as model system. We have extensively studied their protein profiles by ESI LC MS/MS approach, integrated the data with transcriptomics studies as well as with proteomics studies from other laboratories. We have thus identified more than 2,000 proteins expressed in stem cells with high confidence. Pathway analysis of these proteins was carried out using KEGG, IPA, GenMAPP and their gene ontology classification revealed transcription regulators, signal transducers, cell cycle and differentiation molecules along with other general classes of proteins. Based on the specific proteins expressed, putative regulatory pathways operational in stem cells could be constructed. Such information on protein expression and plausible biochemical pathways and networks operational in the stem cells and
doi:10.1007/s11568-009-9091-8 pmid:19444648 pmcid:PMC2694876 fatcat:fqidbx6kjzcvtmbhqhy5n7mozq