Comparative Proteomics Research Of Colorectal Cancer Sera And Function Research Of Characteristic Peptide

The comparative proteomics of many tumor had been progressed by using SELDI-TOF-MS (surface enhanced laser desorption/ionization time of flight mass spectrometry) combined with solid-phase surface laminar analysis proteinchip, which included tumor origined from endoderm (colorectal cancer[1,2,20,21], gastric cancer[3], hepatoma[4], esophageal carcinoma[5], pulmonary carcinoma[6]), breast cancer[7] and ovarian cancer[8]. The specific biomarkers of those tumors were discovered, which were up-regulated comparative with normal sera. Good diagnosis results were gained by using those biomarkers. But there was not tandem mass spectrometry and there was not enough resolution to complete peptide mass fingerprinting (PMF) according to SELDI technology. As a result, the biomarkers discovered during the prior research could be not indentified. And the function of those biomarkers could not be researched without sequence information.Based on the accumulative sera sample during the prior research, the comparative proteomics of colorectal cancer had been progressed by using MALDI -TOF/TOF-MS (Matrix Assisted Laser Desorption/Ionization time of flight tandem mass spectrometry) combined with liquid phase surface laminar analysis technology, which was Immobilized Metal ion Affinity Chromatography Magnetic Bead (IMAC MB).30 cases of colorectal cancer patients and 30 cases of healthy people were randomly sampled from the sera library which was established by our lab. The ratio of male to female was 1:1. The age distribution was between 40 and 60. The median age of male and female were 53 and 51, respectively. And all the colorectal cancer preoperative patients were affirmed by pathologic diagnosis. The colorectal cancer group included 1 case of Dukes A stage, 29 cases of Dukes BCD stage. The colorectal cancer group and healthy people group had the similar exposure history. And the influencing sera protein content relative diseases were excluded. The doubled blind test group included 50 cases of colorectal cancer and 50 cases healthy people, which was used to confirm the colorectal cancer biomarkers. The data acquired from MALDI-TOF/TOF-MS was analyzed by using genetic algorithm (GA), quick classifier, support vector machine, supervised neural network algorithm (SNN) biostatistics methods. The 46 sera protein or peptide content difference between colorectal cancer and healthy people was significant (P<0.05). The pattern established by the supervised neural network algorithm method was optima pattern confirmed by double blind test with 100% specificity and 100% sensitivity. According to weighting score and receiver operating characteristic curve (ROC curve), the diagnosis value of the peptide of 1466.9Da was highest in the supervised neural network algorithm pattern. Comparatively with the prior results by using SELDI-TOF-MS combined with IMAC proteinchip, there was no same pattern. The content of 1466.9Da peptide in the colorectal cancer patients'sera was higher than healthy people sera, and that peptide was denominated colorectal cancer characteristic peptide (CCCP) by our lab.The tandem mass spectrum data of CCCP was searched in the MASCOT database, but there was no result. The sequence of CCCP was inferred according the rule of fragment ions by using DeNoVo method. A homology search of that peptide using the BlastP Non-redundant protein sequences (nr) in the GeneBank databases indicated that the CCCP located in the position between 42th and 53th amino acids of ROMO1 (reactive oxygen species modulator 1) and in the long arm of 20th chromosome. The M/Z of ROMO1 is same to M/Z of the colorectal cancer biomarker which was discovered by SELDI combined with IMAC proteinchip. They were both 8.9kD. But the sequence of biomarker is unknown, so the relationship between them was not clear. The CCCP was maybe product of metabolism of ROMO1, or it could be the alternative splicing of ROMO1 mRNA. Many species had the same sequence of CCCP, so the CCCP possessed the high evolution conservation. That indicated the function of CCCP was perhaps important during the process of physiology and pathophysiology procedure. By bioinformatics analysis, CCCP possessed the transmembrane zone or the hydrophilia and lipophilia characteristics. Wallace WE[40] and Ressom HW[41] discovered that the content ofα-defensin-1,2,3 in the colorectal cancer patients sera and tiusse was higher than the healthy people. So the hypothesis was that CCCP was not only the colorectal cancer biomarker, but also had the anti-bacterial activity.To indentify the antibacterial activity of CCCP, the study of quantitive antibacterial activities against the laboratory bacteria was designed. The antibacterial spectrum of CCCP was researched, and the results represented that CCCP had the antibacterial activity against both G+ bacteria and G- bacteria. It is more significant that CCCP had the antibacterial activity against the AmpR bacteria.In this study, the in-vitro antibacterial mechanisms of the referred peptide were preliminarily investigated. CCCP was incubated with the pTAT-EGFP E.Coli BL-21, and FCM analysis of the resuspension of bacteria precipitation was used to count the bacteria ratio dyed by PI and Western Blot of the supernatant was detected whether the GFP of target bacteria was leaked out. Both methods co-analyzed the membrane integrity of target bacteria. All the results had indicated the CCCP might kill target bacteria by the way destroying the membrane integrity leading content of the target bacteria to leak out.The side-effect of CCCP, hemolytic effect, was preliminarily evaluated. In this study, in-vitro hemolytic effects were evaluated by detecting the OD570 value of the supernatant after CCCP and RBC were incubated. The results demonstrated CCCP had no in-vitro hemolytic effects to human erythrocyteAll the investigation indicated that:1. Compared with several modeling method, the optima pattern was inditified, which was established by supervised neural network algorithm method;2. CCCP was discovered, whose content in colorectal cancer patients sera was higher than the healthy people. And the specificity and sensivity were both 100%, by using CCCP diagnosing colorectal cancer;3. The amino acids sequence of CCCP was indentified by using DeNoVo analysis;4. A homology search of this peptide using the BlastP Non-redundant protein sequences (nr) in the GeneBank databases indicated many species had the same sequence of CCCP and CCCP possessed the evolution conservation. There was no report of the function of CCCP. That also indicated that the function of CCCP was perhaps important during the process of physiology and pathophysiology;5. By bioinformatics analysis, CCCP possessed the transmembrane zone and the hydrophilia and lipophilia characteristics.6. Antibacterial activity could be found only when the concentration of the CCCP reached the threshold dosage;7. CCCP had the antibacterial activity against both G+ bacteria and G- bacteria. It is more significant that CCCP had the antibacterial activity against the AmpR bacteria;8. CCCP might kill target bacteria by the way destroying the membrane integrity;9. CCCP had not in-vitro hemolytic effects to human erythrocyte.