No description available.

No description available.

No description available.

Currently, the detection/quantitation of biomolecules using Inductively Coupled Plasma Mass Spectrometry (ICR-MS) is focused on heteroatom containing (e.g. S. P, Se, Cu) compounds. This limits the scope of ICP-MS as a routine analytical tool in the bioanalytical sciences. To increase this scope novel strategies to incorporate elemental labels have been developed. Chemical derivatisation is one such strategy. Cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA), a bifunctional chelating agent (BFCA), is reacted with N-terminal amines and is subsequently chelated with trivalent metals. This allows the detection of peptides that contained no heteroatom using liquid chromatography (LC)-ICP-MS. The derivatisation efficiency, optimised using LC-UV/vis detection, is approximately 99%. The derivatisation and chelation reactions products are characterised using LC-electrospray ionisation tandem mass spectrometry (ESI-MS-MS). The labelling procedure allowed the relative quantitation of peptides, by differential isotopic labelling. Two individually derivatised peptide samples are chelated with natural isotopic abundance and isotopically enriched 151Eu respectively. The Eu labelled peptides were combined and analysed by LC-ICP-MS. The resulting 153EU:151EU ratio, measured using the pseudo steady state approach for transient signals; is then used to calculate the original peptide proportions using a modified isotope dilution equation. The ICP-MS measured peptide ratio was within 2.8% of the theoretical peptide ratio. The absolute detection limit for the relative quantitation of Eu labelled bradykinin was 5 pmol, which is comparable to current ESI-MS methods. When cDTPA derivatisation was applied to a complex sample multiple by-products were observed in the LC-UV/vis chromatogram. However, the corresponding LC-ICP-MS chromatogram suggested that only singly derivatised peptides were chelated with the metal. To overcome by-product formation monoreactive BFCAs, namely isothiocyanate-benzyl- EDTA (SCN-Bz-EDTA) and isothiocyanate-benzyl-DTPA (SCN-Bz-DTPA). were evaluated as derivatisation reagents. Solid phase analytical derivatisation (SPAD) and solid phase extraction (SPE) were also evaluated for speed and ease of use. The monoreactive nature of SCN-Bz-EDTA and SCN-Bz-DTPA gave singly derivatised peptides when applied to complex samples. However, the resulting derivatisation efficiency was low and no significant improvement in efficiency was noted when SPAD was used. SCN-Bz-EDTA derivatised peptides could not be chelated to trivalent metals, whilst chelation occurred with SCN-Bz-DTPA derivatised peptides. Eu labelled peptides were isolated and selectively extracted by SPE for relative quantitation by LC-ICP-MS. However, due to the instability of the isothiocyanate reagent and instrumental effects the relative error on the measured peptide ratio was greater than 45% when compared to the theoretical ratio. Although the peptide ratio obtained using the SPE method agreed with the ratio from the non extracted sample.

The identification of proteins and peptides has become one of the most common applications in biological mass spectrometry. A primary aim of analysts in protein and peptide analysis is to reduce the complexity of sample mixtures in order to enhance sensitivity and selectivity. The research reported in this thesis describes the development of mass spectrometry-based methods for the separation and analysis of peptide mixtures for rapid, high-throughput proteomic analysis.

Fluorescent techniques are amongst the methods used to study the self assembly of proteins into oligomers, however, most of the reports in literature use extrinsic fluorophores to obtain information on the peptide system. The goal of this thesis is to demonstrate how the intrinsic Tyrosine fluorescence of the β-amyloid peptide can be used to monitor its own aggregation with, therefore, minimal interference on the peptide's native structure, its biochemistry and its spontaneous process of aggregation. Firstly, it is shown that the fluorescence of amino acid Tyrosine, naturally present in wild type β-amyloid, responds to the spontaneous aggregation of the peptide. This is achieved by performing time-correlated single-photon counting experiments during the process of the peptide's aggregation into amyloid fibrils. Through comparison with the well established Thioflavin T assay is also demonstrated that Tyrosine decay responds to changes caused by peptide aggregation well before the appearance of the characteristic β- sheet structures present in the fibrils. Then the use of β-amyloid's intrinsic Tyrosine amino acid as a sensor for the pre- brillar stages of aggregation is further tested and researched through a series of experiments with different initial β-amyloid concentrations. Patterns of consistent behaviour are found confirming that Tyrosine can act as a sensor for the formation of oligomers and kinetic information about the oligomerisation process is retrieved. Using this approach a comparison between the oligomerisation kinetics of the two most common variants of β-amyloid is performed. In the process of studying Tyrosine response to the peptide aggregation the accuracy of the discrete exponential decay model used to describe the decays is debated. A model-free analysis is used to study Tyrosine decay photophysics in the β-amyloid peptides throughout the process of aggregation. It is found that Tyrosine decay is a composition of four discrete decay components suggesting the existence of four rotameric forms of the amino acid in the Fluorescent techniques are amongst the methods used to study the self assembly of proteins into oligomers, however, most of the reports in literature use extrinsic uorophores to obtain information on the peptide system. The goal of this thesis is to demonstrate how the intrinsic Tyrosine uorescence of the β-amyloid peptide can be used to monitor its own aggregation with, therefore, minimal interference on the peptide's native structure, its biochemistry and its spontaneous process of aggregation. Firstly, it is shown that the uorescence of amino acid Tyrosine, naturally present in wild type β-amyloid, responds to the spontaneous aggregation of the peptide. This is achieved by performing time-correlated single-photon counting experiments during the process of the peptide's aggregation into amyloid brils. Through comparison with the well established Thioavin T assay is also demonstrated that Tyrosine decay responds to changes caused by peptide aggregation well before the appearance of the characteristic - sheet structures present in the brils. Then the use of β-amyloid's intrinsic Tyrosine amino acid as a sensor for the pre- brillar stages of aggregation is further tested and researched through a series of experiments with different initial β-amyloid concentrations. Patterns of consistent behaviour are found con rming that Tyrosine can act as a sensor for the formation of oligomers and kinetic information about the oligomerisation process is retrieved. Using this approach a comparison between the oligomerisation kinetics of the two most common variants of β-amyloid is performed. In the process of studying Tyrosine response to the peptide aggregation the accuracy of the discrete exponential decay model used to describe the decays is debated. A model-free analysis is used to study Tyrosine decay photophysics in the β-amyloid peptides throughout the process of aggregation. It is found that Tyrosine decay is a composition of four discrete decay components suggesting the existence of four rotameric forms of the amino acid in the -amyloid peptides. The ndings are further corroborated by molecular dynamic simulations, breaking with the traditional model of three rotameric forms for the uorescent amino acids in protein chains. Finally, the sensing method is used to study the inuence of external uorophores, both associating and covalently bound, on the process of oligomerisation. β-amyloid peptides. The findings are further corroborated by molecular dynamic simulations, breaking with the traditional model of three rotameric forms for the fluorescent amino acids in protein chains. Finally, the sensing method is used to study the inuence of external uorophores, both associating and covalently bound, on the process of oligomerisation.

No description available.

B-cell tropin (BCT) is a pro-opiomelanocortin-derived peptide which is released from the pituitary neurointermediate lobe (NIL) of rodents and other mammals. It has been shown to act as a potent insulin secretagogue, has lipogenic properties both in vitro and in vivo, and its sequence has been characterized as corresponding to ACTH22-39. Perifusate of NIL can be fractionated by reverse phase HPLC into two peaks, one containing CLIP (ACTH 18-39) -related, and the other BCT-related material. BCT levels have been found to be elevated in various obese/diabetic animal models, and this, coupled with the peptide's lipogenic properties, suggests that it may have a role in the obesity commonly associated with the human disease, type II diabetes. The aim of the work described in this thesis was to assess the factors controlling the release of BCT from the pituitary in the normal and obese diabetic state. The obese (oblob) mouse and its lean (+/+) counterpart were used as the animal models for these metabolic conditions.

This thesis describes the investigation of methodologies towards the synthesis and purification of 5-, 6- and 7- membered guanidine heterocycles. Methods include intemlolecular epoxide ring opening reactions using guanidine and the intramolecular cyclization reactions of N-Boc-allylguanidines and N-Boc-homoallylguanidines in both epoxide ring opening reactions and palladium mediated cyclisations. The possible utility of these heterocycles as glycosidase inhibitors was investigated using enzymatic assays.

In recent years, several studies have suggested a role for PKMs, an atypical isoform of PKC, in memory consolidation and long-term potentiation (LTP). Some evidence in support of this theory has come to light, in the form of electrophysiological and behavioural studies, using inhibition, knockdown, perfusion of the molecule itself and overexpression. Exogenously applying the PKM~ to systems leads to potentiation. Conversely, application of Zeta Inhibitory Peptide (ZIP), a peptide sequence purported to inhibit PKMs, consistently reverses LTP and in most cases, erases memories. To date, however, studies on PKMs have focused on functional aspects of PKMs activity (i.e. behaviour and synaptic function) while the molecular events underlying these processes are poorly understood and there are currently few published studies closely examining such details. The experiments performed for this thesis attempt to better understand the molecular effects of ZIP in AMPAR trafficking, and result in some novel findings on the subunitspecific effects, the mechanisms involved, as well as some data showing effects of different concentration of inhibitor. Experiments performed for this project showed that ZIP causes an increase in internalisation of GluAl and GluA2 subunits, and a decrease in surface GluA2, but not GluAl. Previously it had been reported that ZIP leads to a decrease in synaptic function and loss of memory, and this effect can be blocked by preventing GluA2 internalisation. These data are therefore consistent with published studies and also show a new effect regarding the trafficking of the GluAl subunit. It was also shown that PICKl interaction with GluA2, as well as actin depolymerisation are required for the increase in internalization (Rocca et al., 2008) and that PICKl interaction with GluA2 may be increased in the presence of ZIP. These new data support the theory NSF and PICKl are involved in the trafficking activity of PKMs on GluA2 (Yao et al., 2008), and that the loss of synaptic function observed in the presence of ZIP requires PICKl (Yao et al., 2008).