Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

Novel Mass Spectrometry-based Epitope Mapping Procedures of Autoantigens

Preventive medicine for patients is to be realized from monitoring and management of early stage disease rather than from late stage treatment. Among the strategies of preventive medicine is the detection of prognostic and diagnostic protein signatures, in particular epitopes against which autoantibodies in blood plasma are directed during the progression of the disease. Recognizing such diseasespecific antibodies in patient screenings requires knowledge of the respective epitopes. In this work, novel mass spectrometry-based methodologies for epitope mapping of autoantigens are described

New Techniques for the Qualitative and Quantitative Measurement of Naturally-Ocurring Gonadotropin-Releasing Hormone Analogues by Mass Spectrometry

GnRH peptides have been discovered in a wide variety of vertebrate and invertebrate organisms, and work is ongoing to characterize additional unique isoforms. This dissertation describes the investigation of reversed-phase chromatographic and mass spectrometric behavior of GnRH peptides, the development and application of an LC-MS/MS method for qualitative identification of GnRH peptides, and the comprehensive validation of an LC-MS/MS method for simultaneous, quantitative measurement of hydroxyproline9GnRH (Hyp9GnRH) and mammalian GnRH (mGnRH) in rat brain tissues. Chromatographic and mass spectrometric behavior of GnRH isoforms was characterized for six GnRH model peptides. Using reversed-phase high performance liquid chromatography (HPLC), nearly complete separation of the model GnRH peptides was achieved. Evaluation of electrospray source conditions indicated that certain parameters can be adjusted to affect the abundance of selected charge states and improve response. Using the conditions found to be optimal for GnRH peptides in general, a method was developed to facilitate characterization of novel GnRH isoforms or confirm the identity of known isoforms. Fragmentation patterns for six model GnRH isoforms were examined to determine what portion of the primary sequence could be elucidated by de novo sequencing, and a simple solid phase extraction protocol was developed to isolate the model GnRH compounds from tissue samples. Application of the method to rat brain samples resulted in successful isolation and structural confirmation of hydroxyproline9GnRH and mammalian GnRH. A quantitative method for the determination of concentrations of hydroxyproline9GnRH and mammalian GnRH in rat brain tissue was developed and rigorously validated. Guinea pig brains were found to be a suitable substitute matrix for rat brains, and accuracy and precision were determined after four validation runs. Stability of both peptides in samples over long-term storage and under experimental conditions were evaluated, and the LC-MS/MS method was compared to an enzyme-linked immunoassay. Thirty-one brains from Sprague-Dawley rats were analyzed using the LC-MS/MS procedure and compared to published results for Hyp9GnRH and mGnRH

A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels

A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659 Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH2. This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels

Glycoproteomic research using mass spectrometry

The development of problem-specific mass spectrometric (MS) glycoproteomic strategies has allowed the discovery of previously unknown protein glycosylation in both eukaryotic and prokaryotic organisms. The research in this thesis focuses on the identification and structural characterisation of novel glycan structures in ADAMTS13 and Clostridium difficile. ADAMTS13 is a large multi-domain protein which regulates thrombogenesis by cleavage of the adhesive blood glycoprotein, VWF, so generating smaller less thrombogenic fragments. Glycoproteomic strategies were employed to investigate a secretion-enhancing mutant by comparing the O-glycome of wild type (WT) with synonymous substitution, P118P, and a non-synonymous control, P118F. Identical post-translational modifications (PTMs) but several novel PTMs were discovered in ADAMTS13 including TSR1 O-glycosylation, C-mannosylation of W387 and DiSialyl Core-1 O-glycosylation of S1170. Clostridium difficile is one of the main organisms responsible for morbidity in hospitalised patients, and is the etiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis. The C.difficile cell wall is surrounded by an S-layer composed of two proteins, high molecular weight (HMW) and low molecular weight (LMW) SLPs. 12 slpA gene cassettes have been recently described and cassette-11 carries an insert containing 19 ORFs. Combining different biochemical and ES- and MALDI-MS approaches, including the ETD technique, with genetic experiments, it was demonstrated that LMW SLP in strain Ox247 is glycosylated with a surprisingly large linear pentose-branched oligosaccharide of more than forty sugar residues, and a collaborative NMR study suggests a Phospho- and Acetyl- substituted non-reducing terminal rhamnose. Analysing different C.difficile hypervirulent strains, novel flagellar sulphonated peptidylamido-glycan structures not previously observed in sugar or amino acid chemistry were identified. High resolution mass measurement and negative-ion nanospray MS/MS of cone-voltage-induced fragment ions were crucial in allowing the discovery of a unique terminal Taurine (aminoethyl-sulphonic acid) peptidylamidoglycan unit which could provide a novel strategy to escape the immune system, by the C.difficile becoming more virulent.Open Acces

Characterization of the Extracellular Proteome of a Natural Microbial Community with an Integrated Mass Spectrometric / Bioinformatic Approach

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function. To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH \u3c 1) and hot (temperature \u3e 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment. The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the \u3e 100,000 mass spectra collected during an MS experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth

N-terminal Imine Derivatization for Enhanced De Novo Peptide Sequencing: A Study of the Fragmentation Pattern Generated from CID of Peptide-Imines

In this work, the fragmentation pattern derived from model peptides derivatized to create N-terminal imines (Schiff bases) were evaluated. Collision-induced dissociation of the protonated peptide-imines ([M+H]+) generally produced complete series of the sequence informative an and bn ions, now undoubtedly characteristic of the peptide ion species. A novel product ion was also observed, denoted the yǂ ion, determined by IRMPD spectroscopy and density functional theory to be generated from the elimination of the N-terminal amino acid residue despite the N-terminal modification. It was concluded the pathway involved a nucleophilic attack by an amide nitrogen and the possible formation of an imidazole-4-one intermediate, which collapses to generate a truncated, protonated peptide-imine with a conserved primary sequence. N-terminal imine-modification was also observed to eliminate sequence scrambling events, presumably by eliminating the macrocyclic b ion mechanism implicated in the sequence rearrangements. Additionally, the CID mass spectra of Ag-cationized imine-modified peptides were obtained. An apparent even-electron, [M-H]+ peptide ion was observed, determined to be generated by the loss of AgH. The hydrogen abstraction was explicitly identified to originate from the imine-carbon of the argentinated modified peptide. CID of the [M–H]+ ions generated sequence ions analogous to those produced from the [M+H]+ species of imine-modified peptides, however less extensively