Analytical methods for the identification of MHC restricted peptides by mass spectrometry

Clayton, J.E., 2006. Analytical methods for the identification of MHC restricted peptides by mass spectrometry. PhD, Nottingham Trent University.

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Abstract

HPLC and mass spectrometry are versatile analytical techniques which have been widely applied to the analysis of naturally occurring proteins and peptides. Mass spectrometric conditions have been investigated for the analysis of MHC class I and II restricted peptides. A micro-electrospray ionisation source has been constructed for coupling with a capillary HPLC system and investigations have been performed to increase the sample loading volume onto the system.

A robust method has been developed for the clean-up and identification of MHC class I and class II restricted peptides from surface eluted cells by mass spectrometric techniques. The procedure utilises solid phase extraction to desalt and concentrate the cell surface eluates followed by fractionation using strong cation exchange chromatography and analysis by on-line capillary HPLC/μESI/MS/MS using a quadrupole ion trap. The method has been shown to recover model peptides at an immunologically significant level. The procedure has been applied to the analysis of cell surface eluates of patients suffering from chronic myeloid leukaemia and to eluates from k562 transfected cell lines. The purpose of these analyses was to acertain if a selection of HLA.A2 predicted peptides, originating from the bcr/abl fusion protein, are expressed on the surface of leukaemic cells. The clean-up method has also been applied to the identification of the class II invariant chain protein (CLIP) from immunopurified and cell surface eluted class II molecules.

Immobilised metal ion affinity chromatography (IMAC) also known as metal chelate chromatography has been shown to be a powerful tool for the purification of proteins. Using chelated and immobilised copper ions, conditions have been investigated for the selective and non-selective binding of peptides. A histidine selective method has been developed, using a HEPES buffer at pH 7.2, which is ~90% selective. This method has been applied to the purification of cellular extracts and CML patient eluates prior to analysis by HPLC/MS/MS. The method has been shown to remain highly selective when subjected to these complex samples.

Item Type:	Thesis
Creators:	Clayton, J.E.
Date:	2006
ISBN:	9781369313796
Identifiers:	Number Type PQ10183098 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	17 Sep 2020 14:12
Last Modified:	28 Jun 2023 10:09
URI:	https://irep.ntu.ac.uk/id/eprint/40766

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