The glycoproteome is an intricate and diverse component of a cell, and it plays a key role in the definition of the interface between that cell and the rest of its world. Methods for studying the glycoproteome have been developed for released glycan glycomics and site-localized bottom-up glycoproteomics using liquid chromatography-coupled mass spectrometry and tandem mass spectrometry (LC-MS/MS), which is itself a complex problem. Algorithms for interpreting these data are necessary to be able to extract biologically meaningful information in a high throughput, automated context. Several existing solutions have been proposed but may be found lacking for larger glycopeptides, for complex samples, different experimental conditions, different instrument vendors, or even because they simply ignore fundamentals of glycobiology. I present a series of open algorithms that approach the problem from an instrument vendor neutral, cross-platform fashion to address these challenges, and integrate key concepts from the underlying biochemical context into the interpretation process. In this work, I created a suite of deisotoping and charge state deconvolution algorithms for processing raw mass spectra at an LC scale from a variety of instrument types. These tools performed better than previously published algorithms by enforcing the underlying chemical model more strictly, while maintaining a higher degree of signal fidelity. From this summarized, vendor-normalized data, I composed a set of algorithms for interpreting glycan profiling experiments that can be used to quantify glycan expression. From this I constructed a graphical method to model the active biosynthetic pathways of the sample glycome and dig deeper into those signals than would be possible from the raw data alone. Lastly, I created a glycopeptide database search engine from these components which is capable of identifying the widest array of glycosylation types available, and demonstrate a learning algorithm which can be used to tune the model to better understand the process of glycopeptide fragmentation under specific experimental conditions to outperform a simpler model by between 10% and 15%. This approach can be further augmented with sample-wide or site-specific glycome models to increase depth-of-coverage for glycoforms consistent with prior beliefs.

Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

No description available.

Ip Wai-Ho Herman. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 87-90). / Abstracts in English and Chinese. / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Table of Content --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Fourier transform ion cyclotron resonance mass spectrometry (FRICR) --- p.2 / Chapter 1.1.1 --- History of FTICR --- p.2 / Chapter 1.1.2 --- Theory of FTICR --- p.4 / Chapter 1.1.3 --- FTICR with electrospray ionization source --- p.10 / Chapter 1.2 --- Tandem mass spectrometry --- p.11 / Chapter 1.2.1 --- Introduction --- p.11 / Chapter 1.2.2 --- Collision-induced dissociation --- p.12 / Chapter 1.2.3 --- Surface-induced dissociation --- p.12 / Chapter 1.2.4 --- Photodissociation --- p.13 / Chapter 1.2.5 --- Blackbody infrared radiative dissociation (BIRD) --- p.13 / Chapter 1.3 --- Electron capture dissociation --- p.13 / Chapter 1.4 --- Recent advances in ECD experiments --- p.15 / Chapter 1.5 --- Outline the present work --- p.17 / Chapter 2. --- EXPERIMENTAL AND INSTRUMENTATION --- p.18 / Chapter 2.1 --- Instrumentation --- p.19 / Chapter 2.1.1 --- Fourier-transform ion cyclotron resonance mass spectrometer --- p.19 / Chapter 2.1.2 --- Vacuum system --- p.19 / Chapter 2.1.3 --- Electrospray ionization source --- p.23 / Chapter 2.1.4 --- Electrostatic ion focusing system --- p.26 / Chapter 2.1.5 --- Infinity cell --- p.28 / Chapter 2.1.6 --- Electron emission source --- p.29 / Chapter 2.1.7 --- Data acquisition system --- p.32 / Chapter 2.2 --- Experimental --- p.32 / Chapter 2.2.1 --- Simple acquisition pulse program --- p.32 / Chapter 2.2.2 --- ECD pulse program with/without collision cooling --- p.35 / Chapter 3. --- OPTIMIZATION OF EXPERIMENTAL PARAMETERS FOR ELECTRON CAPTURE DISSOCIATION --- p.38 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Experimental --- p.40 / Chapter 3.2.1 --- Materials --- p.40 / Chapter 3.2.2 --- Sample preparation --- p.41 / Chapter 3.2.3 --- Instrumentation --- p.41 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- Benchmark conditions --- p.42 / Chapter 3.3.2 --- Effect of the filament heating currents (If) --- p.45 / Chapter 3.3.3 --- Effect of the average filament bias voltages (Vf) --- p.49 / Chapter 3.3.4 --- Effect of the electron irradiation time (te) --- p.53 / Chapter 3.3.5 --- Reduction of the fragment ions intensity --- p.56 / Chapter 3.3.6 --- Effect of the trapping potentials --- p.60 / Chapter 3.4 --- Conclusions --- p.62 / Chapter 4. --- ENHANCEMENT ON ELECTRON CAPTURE DISSOCIATION EFFICIENCY --- p.63 / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- Experimental --- p.65 / Chapter 4.2.1 --- Materials --- p.65 / Chapter 4.2.2 --- Sample preparation --- p.65 / Chapter 4.2.3 --- Instrumentation --- p.65 / Chapter 4.3 --- Results and discussion --- p.66 / Chapter 4.3.1 --- Effect of the filament position --- p.66 / Chapter 4.3.2 --- Effect of the collision gas pressure --- p.68 / Chapter 4.3.3 --- Effect of the collision gas --- p.73 / Chapter 4.3.4 --- Effect of the electron irradiation at different pulse gas interval --- p.75 / Chapter 4.3.5 --- Effect of the multiple electron irradiation --- p.76 / Chapter 4.3.6 --- Optimized Conditions --- p.79 / Chapter 4.4 --- Conclusions --- p.84 / Chapter 5. --- CONCLUSIONS --- p.85 / Chapter 5.1 --- Conclusions --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.91 / Appendix A Simple pulse sequence program for ESI FTICR-MS experiments --- p.91 / Appendix B Pulse sequence program for ESI FTICR-MS electron capture dissociation --- p.95 / Appendix C Pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with collision cooling --- p.99 / Appendix D Modified pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with a time lag between collision cooling and electron irradiation. --- p.104 / Appendix E Modified pulse sequence program for ESI FTICR-MS electron capture dissociation experiments with multiple irradiation --- p.109

Redox potential, a measure of how oxidising or reducing an environment is, is tightly regulated by cells to minimise detrimental chemical oxidation and reduction reactions. In proteins, it is the sulfur containing cysteine residues that can be post-translationally modified through specific redox reactions, for example, the formation of disulfide bonds between cysteine residues can be crucial to protein structure. It has recently been hypothesised that signalling pathways utilising redox regulated proteins may be arranged into electrochemical series. The characterisation of the redox properties of specific cysteine residues in proteins has proven difficult using traditional redox characterisation methods such as cyclic voltammetry. A number of biochemical methods have been developed for studying the effect of the redox environment on proteins, many making use of mass spectrometry and allowing for localisation of the site of the modification to specific cysteine residues. However, fewer methods have been reported that facilitate accurate quantification for the determination of the mid-point potential of these redox regulated cysteine residues. Here, a differential labelling protocol using high resolution mass spectrometry techniques for the study of redox chemistry of cysteine residues in proteins will be reported. The protocol exploits the novel chemistry of thiol groups for specific alkylation and allows for both qualitative and quantitative experiments. Thioredoxin-1 from E. coli and human systems was used as a model protein and a novel disulfide bond was characterised. The reducing potential of the active site cysteine residues of human thioredoxin were found to be very similar to those of the E. coli proteoform, -276 ± 1 and -281.4 ± 0.3 mV respectively. The remaining three cysteine residues of human thioredoxin were found to be regulated at more oxidising potentials. The protocol developed was applied to a protein from the cell death pathway of apoptosis; human caspase-3 is an executioner protease from the caspase cascade. Caspase-3 was found to contain three redox sensitive cysteine residues. The catalytically active cysteine residue was redox regulated via two mechanisms, glutathionylation and disulfide bond formation. One of these mechanisms gives the active site cysteine residue a calculated reducing potential of -165 ± 6 mV supporting the correlation between caspase-3 activity and its observed role in the apoptotic pathway but not in necrotic cell death.

No description available.

No description available.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Aug 16