Phosphorylation is a key regulator of protein function and activity, and aberrant kinase activity is implicated in a wide range of malignancies, of which the bcr:abl fusion kinase found in chronic myeloid leukaemia is a classic example. As phosphopeptides are known to be presented by both the MHC class-I and class- II pathways, against which specific CD4+ and CD8+ T cell responses may be generated, study of MHC-presented phosphopeptides may reveal unique cancer antigens with direct links to the neoplastic state. Mild acid cell-surface elution is a rapid and effective method for MHC class-I peptide capture, though complicated by contamination with non-MHC peptides and poor downstream compatibility, especially with IMAC, a popular method for phosphopeptide enrichment. As an alternative to the citrate-phosphate elution buffer, a TMA-formate elution buffer is proposed. This was developed for IMAC compatibility, and osmotically balanced and supplemented to minimise cell lysis, (assessed by several assays) and used with a pH 5.5 prewash to reduce non- MHC peptide contamination. MALDI-MS/MS of MHC class-I peptides from K562- A3 cells found a sequence with high homology to a known cancer antigen as the common peak for both citrate-phosphate and TMA-formate eluted cells. Currently there are no published mechanisms for cell-surface elution of MHC class-II peptides (immunoprecipitation is widely used), though previous work at NTU led to the development of an IMAC compatible MHC class-II protocol. This was also subjected to supplementation and optimisation, reducing cell death to a level corresponding to that of the widely accepted citrate-phosphate class-I protocol. Various chromatographic approaches were tested for phosphopeptide retention. Fe3+ IMAC remains optimal; methods were adjusted to increase peak fraction concentration (assessed by a modification the BCA protein assay improving suitability for peptides). Though further method development may be required to optimise mass spectrometry, a number of phosphopeptides were found in both MHC class-I and class-II eluates, many with known links to malignancy. It is hoped that these improved methods will be of use in the ongoing search for novel cancer antigens.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:629238 |
| Date | January 2011 |
| Creators | Kapoor, K. N. |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://irep.ntu.ac.uk/id/eprint/162/ |
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