Year

2010

Degree Name

Doctor of Philosophy

Department

School of Chemistry - Faculty of Science

Abstract

The majority of naturally occurring proteins are modified in some manner, with many biological systems requiring these modifications in order to function properly. Acylation is one such type of modification. For example, the neuropeptide ghrelin, which plays a critical role in appetite stimulation, is octanoylated on its ser-3 residue. Many proteins are palmitoylated at one or more cysteine residues, with the lipid moiety essential for membrane binding. It is likely that many biological systems rely on protein acylations, and it would be beneficial to develop techniques that allow a facile detection of naturally occurring acylations.

Synthetically acylated peptides and proteins (a class of monodisperse polymer) have many potential uses. Synthetically acylated peptides and proteins are being developed for use as therapeutic agents and also as chemical standards. Synthetically acylated peptides, for example, have the potential for use as vaccines for diseases such as hepatitis and HIV. The ability to successfully characterise these types of semi-synthetic molecules is imperative in their development process. In this work, mass spectrometry is explored as a means of analysing acylated peptides and proteins.

A number of synthetically acylated peptides were examined using a range of mass spectrometry techniques in order to identify characteristic fragmentations. Acylated peptides were fragmented using electrospray ionisation or matrix-assisted laser desorption/ionisation combined with collisionally induced dissociation tandem mass spectrometry, and also using matrix-assisted laser desorption/ionisation post source decay mass spectrometry. Acylated peptides were observed to fragment in a similar manner to their unacylated counterparts, with the degree of fragmentation observed dependant on the length of the acyl chain (i.e. higher collision energies were required to illicit the same degree of fragmentation with increasing chain length). The presence of an acylation on an N-terminal amino acid allowed the formation of a b1 product ion, not normally observed in the spectrum of unmodified peptides. A number of useful marker ions for acylation at serine, tyrosine and cysteine residues were observed, including, acyl carbenium ions and acylated immonium ions. The neutral loss of the acyl moiety was also commonly observed. The tandem mass spectrometry conditions required to produce marker ions were explored.

Two lipopeptides, synthesised as by-products in the production of N-terminally acylated HIV protein Nef1-6 (acyl-GGKWSK), were characterised. These lipopeptides were found to be twelve residues long, with an unusual ether-linked kynurenine at position seven, and contained either a stearoyl or oleoyl moiety at their N-terminal glycine residues (ste/ole-GGKWSK-O-kyn-SKWSK). The successful characterisation of these molecules has allowed continued investigations into their use as immunological agents.

A range of novel monodisperse polymers, acylated at their lysine residues, were analysed intact or subsequent to enzymatic digestion, using high performance liquid chromatography and tandem mass spectrometry. Analogous tandem mass spectra were observed irrespective of the acylating agent, however, chromatographic elution times were found to be dependant on the identity of the acyl moiety. Acylated lysine carbenium ions were observed in the tandem mass spectra, 17 u lower in mass than the calculated value for an acylated lysine immonium ion.

This work demonstrates the ability of mass spectrometry to enable the characterization of a wide range of acylated peptides and proteins. The use of a variety of mass spectrometry and commonly employed analysis techniques (such as enzymatic digestion) further aid in the characterisation of acylated peptides and proteins.

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