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Lill, J.R., 2000. Characterisation of MHC class 1 tumour antigens. PhD, Nottingham Trent University.
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Abstract
A protocol has been developed and optimised for the isolation and mass spectrometric characterisation of major histocompatibility complex (MHC) class I tumour associated antigens. The procedure involves the use of mild acid elution for the isolation of cell surface MHC class I associated peptides, through destabilisation of the MHC β2-microglobulin, and protein precipitation with trichloroacetic acid. A two-dimensional chromatographic procedure using cation exchange and microbore reverse-phase high performance liquid chromatography (RP-HPLC) is employed for sample clean-up prior to mass spectrometric analysis. A nano-electrospray ionisation source has been constructed, and mass spectrometric conditions optimised for the detection and characterisation of low concentrations (fmol μl-1) of cell surface MHC class I associated peptides. The protocol has been applied for the characterisation of peptides from transfected cell lines and cells derived from Chronic myeloid leukaemia patients. Peptides derived from the novel BCR-ABL fusion region of the Philadelphia chromosome have been characterised, which are specific to a translocation event unique to these haematopoietic malignancies. The mass spectrometric results correlate with T cell in vitro assays, suggesting that these peptides have potential for future tumour immunotherapeutics.
The formation of transition meta/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for MHC class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Full scan mass spectrometry and tandem mass spectrometry of copper, silver, nickel, or a combination of these transition metals/peptide complex ions provides peptide sequence information and an insight into the peptide chelation sites.
| Item Type: | Thesis | ||||
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| Creators: | Lill, J.R. | ||||
| Date: | 2000 | ||||
| ISBN: | 9781369316186 | ||||
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| Divisions: | Schools > School of Science and Technology | ||||
| Record created by: | Linda Sullivan | ||||
| Date Added: | 25 Sep 2020 13:41 | ||||
| Last Modified: | 23 Aug 2023 13:09 | ||||
| URI: | https://irep.ntu.ac.uk/id/eprint/40944 |
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