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Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase.
In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues.
For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%.
Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out usingBLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%.
Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma.
Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.
© 2008 Ngoka; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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VCU Chemistry Publications
HepG2_1795 unique proteins identified in the MudPIT mass spectra of HepG2 cells. When Bioworks version 3.2 searched the HepG2 MudPIT data against NCBI nr database, 77831 hits were returned. Then, upon post-search processing and validation, only 1795 out of the original hits are confirmed. The post-search processing is based upon the criteria that: only peptides identified as possessing fully tryptic termini (containing up to two missed internal trypsin cleavage sites), with cross-correlation scores (Xcorr) greater than 1.9 for singly charged peptides, 2.3 for doubly charged peptides and 3.75 for triply charged peptides, are used for peptide identification. In addition, the delta-correlation scores (ΔCn) must be greater than 0.1 for peptide identification, and protein probability was set at ≤ 1e-002.
1477-5956-6-29-s2.xls (419 kB)
Human Liver_1819 unique proteins identified in the MudPIT mass spectra of normal human liver tissue. post-search criteria are identical to those of the HepG2 proteome of Additional File 1: Only 1819 validated, unique proteins are retained out of the 79961 original hits of the normal human liver proteome.
1477-5956-6-29-s3.tiff (5601 kB)
MudPIT mass spectra of HepG2 cells (left) and normal human liver tissue (right). There are twenty-four nano-LC/ESI-MS/MS spectra, two of which are 1D nano-LC/ESI-MS/MS. Twenty-two are 2D nano-LC/ESI-MS/MS spectra. Each MudPIT experiment consists of a 12-cycle run in which a 60-minute nano-LC/ESI-MS/MS gradient is run for each of: 1D, 2D, 2D (0 mM NH4COO), 2D (25 mM NH4COO), 2D (50 mM NH4COO), 2D (75 mM NH4COO), 2D (100 mM NH4COO), 2D (150 mM NH4COO), 2D (200 mM NH4COO), 2D (250 mM NH4COO), 2D (300 mM NH4COO) and 2D (500 mM NH4COO).
1477-5956-6-29-s4.xls (411 kB)
HepG2_Sequence Table_BLASTP 2.2.13_ [Nov-27-2005]. The results of a Mapping operation are presented in the form of a Sequence Table, which consists of nine parameters (Headers): Sequence name, Seq description, Length, #hits, Maximum eValue, Similarity mean, number of Ontologies (GOs) found, the GO identification numbers of the found Ontologies, Enzyme (i.e. Enzyme Commission number).
1477-5956-6-29-s5.xls (420 kB)
Human Liver_Sequence Table_BLASTP 2.2.13_ [Nov-27-2005]. Description of Sequence Table same as for Additional file 4.
1477-5956-6-29-s6.xls (402 kB)
HepG2_Sequence Table_BLASTP 2.2.15_ [Oct-15-2006]. Description of Sequence Table same as for Additional file 4.
1477-5956-6-29-s7.xls (401 kB)
Human Liver_Sequence Table_BLASTP 2.2.15_ [Oct-15-2006]. The results of a Mapping operation are presented in the form of a Sequence Table, which consists of nine parameters: Sequence name, Seq description, Length, #hits, Maximum eValue, Similarity mean, number of Ontologies (GOs) found, the GO identification numbers of the found Ontologies, Enzyme (i.e. Enzyme Commission number).