Howell, Steven Alan; (1993) Applications of mass spectrometry in the analysis of proteins, modified peptides and chiral isomers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The current expansion in the application of mass spectrometry (MS) to determine the primary structures of proteins and peptides is reflected in the bulk of this thesis. At the protein level, electrospray and fast atom bombardment (FAB) MS have been integrated with conventional Edman sequencing to establish the sequences of two isoforms of annexin V co-expressed in bovine brain. Using a similar approach the sequences of plastocyanins from the cyanobacterium Scenedesmus obliquus and the blue green alga Anabaena variabilis have also been ascertained. Peptide studies focus on several post translationally modified species. N-terminal acetylation has been proven to exist in thioesterase II from rat mammary gland. The sequences of thymosin β11 and a novel isoform β12 have been determined and shown to be acetylated at their N-termini. The less common modification of N-terminal myristoylation is encountered in the analysis of a pp60src derived peptide. The formation of pyroglutamate has been proven to occur in a novel tripeptide present in human seminal fluid, together with C-terminal amidation. A study is described to identify a similar species present in rabbit prostate complex by FAB sequencing. Phosphorylation is a major post treinslational modification having an essential role in cell regulation, the phosphorylation of a peptide derived from a major substrate of protein kinase C is described. The sequences of peptides derived from bovine heart pyruvate dehydrogenase complex possessing lipoic acid prosthetic modifications have been determined. The final studies of modified peptides focus on the use of FAB-MS to characterise synthetic peptides, with important advantages noted over conventional protocols. The remainder of the thesis concerns a study in the detection of chirality using FAB-MS. The differentiation of dialkyl tartrate enantiomers due to dimer effects is described, however attempts to resolve enantiomers of threonine and serine derivatives were unsuccessful.

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