Aim: To assess SELDI-TOF MS technology as a tool for biomarker discovery in the stool and serum of colorectal cancer patients. Materials and Methods: 1.Initially a technique of analysis was developed and optimised using tumour samples and matched normal mucosa, obtained from the Tayside Tissue Bank. These samples were then analysed using SELDI on a PBS II Protein Chip Mass Spectrometer to identify abundant proteins. 2. A technique of stool preparation and subsequent SELDI analysis was developed and then optimised (CM10 chip at pH4) to allow comparison of faecal samples from cancer and controls. Faecal samples were then collected from cancer patients and controls and analysed. In addition, FOB testing was carried out on all stool samples from cancer and controls and subgroup analysis of spectras was performed controlling for FOB status. 3. A test set of cancer and normal serum samples was used to optimise the method of analysis using 4 different chip surfaces at differing pH. Serum samples were collected from cancer patients and normal controls and were analysed on the H50 chip. Serum was then depleted of major proteins in an attempt to improve the detection of peaks. The mass spectra from each sample type were compared to identify any common protein peaks. Results: 1. Tumour analysis methods were optimised using an initial 4 samples of tumour and normal mucosa. Analysis of 8 further paired samples showed protein peaks at 2826, 3374, 3444, 3489 and 10854 Da which were abundant in tumour and reduced in the normal mucosa. 2. In serum analysis the initial experiment of 10 cancer versus 10 normal revealed 4 peaks on the H50 chip (3479, 3364, 3434, 3700 Da) that had significantly higher mass to charge ratios in cancer. The experiment was repeated on the H50 chip using 92 cancers and 92 controls and 5 different peaks were identified (7901, 8124, 8566, 8799 and 17 409Da) as being significant but these had higher mass to charge ratios in the controls. After depletion of the serum samples of albumin, transferrin, haptoglobin, anti-trypsin, IgG and IgA SELDI-TOF analysis showed a greatly reduced profile that yielded no meaningful spectra. 3. Stool analysis revealed 5 protein peaks (4633, 16511, 33423, 37087 and 47026 Da) in colorectal cancer patients, which were absent in stools from controls with a sensitivity of 83% when using all 5 peaks. Degradation of the spectra was observed after prolonged storage of stool samples. Conclusions: A method of stool analysis has been developed that yielded valid peaks differentiating between cancer and normal, which warrant further research through protein identification. Serum analysis was not reproducible across experiments and depletion of major proteins failed to reveal the sub-proteome raising doubts about whether discovery-based serum proteomics can accurately detect cancer. SELDI-TOF was not able to demonstrate that any of the peaks present in the tumour analysis were present in the stool or the serum samples.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:633764 |
| Date | January 2014 |
| Creators | Henderson, Nikola Alexandra |
| Contributors | Steele, Robert |
| Publisher | University of Dundee |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://discovery.dundee.ac.uk/en/studentTheses/3dcbffa3-7edd-47c9-aca1-e55da49c6288 |
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