Mass spectrometry (MS)based protein and peptide identification has become a solid method in proteomics. In high-throughput proteomics research, the “shotgun method has been widely applied. Database searching is currently the main method of tandem mass spectrometrybased protein identification in shotgun proteomics. The most widely used traditional search engines search for spectra against a database of identified protein sequences. The search engine is evaluated for its efficiency and effectiveness. With the development of proteomics, both the scale and the complexity of the related data are increasing steadily. As a result, the existing search engines face serious challenges. First, the sizes of protein sequence databases are ever increasing. From IPI.Human.v3.22 to IPI.Human.v3.49, the number of protein sequences has increased by nearly one third. Second, the increasing demand of searches against semispecific or nonspecific peptides results in a search space that is approximately 10 to 100 times larger. Finally, posttranslational modifications (PTMs) produce exponentially more modified peptides. The Unimod database (http://www.unimod.org) currently includes more than 1000 types of PTMs. We analyzed the entire identification workflow and discovered three things. First, most search engines spend 50% to 90% of their total time on the scoring module, the most widely used of which is the spectrum dot product (SDP)based scoring module. Second, nearly half of the scoring operations are redundant, which costs more time but does not increase effectiveness. Third, more than half of the spectra cannot be identified via a database search alone, but the identified spectra have a connection with the unidentified ones, which can be clustered by their distances. Based on the above observations, we designed and implemented a new search engine for protein and peptide identification that includes three key modules. First, a parallel index system, based on GPU, organizes the protein database and the spectra with no redundant data, low search computation complexity, and no limitation of the protein database scale. Second, the graphics processing unit (GPU)based SDP module adopts GPUs to accelerate the most time-consuming step in the process. Third, a k-meansbased spectrum-clustering module classifies the unidentified spectra to the identified spectra for further analysis. As general-purpose high-performance parallel hardware, GPUs are promising platforms for the acceleration of database searches in the protein identification process. We designed a parallel index system that accelerated the entire identification process two to five times with no loss of effectiveness, and achieved around 80% linear speedup effect on the cluster. The index system also can be easily adopted by other search engines. We also designed and implemented a parallel SDP-based scoring module on GPUs that exploits the efficient use of GPU registers and shared memory. A single GPU was 30 to 60 times faster than the central processing unit (CPU)based version. We also implemented our algorithm on a GPU cluster and achieved approximately linear acceleration. In addition, a k-meansbased spectrum-clustering module with GPUs can classify the unidentified spectra to the identified spectra at 20 times the speed of the normal k-means spectrum-clustering algorithm.

The use of mass spectrometry for the analysis of petrochemical products and crude oils enables the generation of detailed molecular data essential for chemical characterisation and product development. However, the need for multistage sample preparation techniques can be time consuming and may result in the loss of information. Ambient ionisation in combination with mass spectrometry enables the direct analysis of compounds present on a surface with minimal or no sample preparation. The work presented in this thesis evaluates the application of mass spectrometry (MS) hyphenated with ambient ionisation and ion mobility for the analysis of chemical additives used in lubricant and petrochemical products and also crude oil. A technique called desorption electrospray ionisation (DESI) pioneered the ambient ionisation field. An in-house designed and constructed DESI source has been developed to enable hyphenation of DESI with MS and ion-mobility mass spectrometry (IM-MS) for the interrogation of chemical additives used in lubricant and petrochemical oils directly from multiple surface substrates. The approach has been successfully applied to the analysis of a range of chemical additives as standards and when present in a lubricating oil matrix. Data has also shown that DESI-MS can be used to map additive deposition on a surface. The quantitative capabilities of DESI-MS have been assessed using a lubricant antioxidant additive present in a lubricant oil matrix and deposited on a surface. The DESI-MS method showed good linearity with a limit of detection (LOD) for the antioxidant additive below that used in typical commercial formulations. The use of a suitable internal standard in the DESI-MS analysis has been shown to significantly improve the repeatability of the approach. Hyphenation of DESI with post ionisation separation methods, such as high field asymmetric waveform ion mobility spectrometry (FAIMS), can improve mass spectral response for targeted analytes through selective transmission. The analysis of a series of corrosion inhibitor additives in a base oil matrix has been carried out using electrospray (ESI) and DESI hyphenated with FAIMS-MS. FAIMS selection of target ions improved the sensitivity of ESI and DESI through enhanced analyte transmission and a reduction in the chemical noise resulting from the oil matrix. DESI-FAIMS-MS was shown to improve target analyte response compared to DESI-MS alone using the corrosion inhibitors as model compounds, showing how the combined technique can be used for the rapid analysis of analytes directly from surfaces with no sample preparation or pre concentration. Direct analysis in real time (DART) is an alternative ambient ionisation approach to DESI. The use of DART-MS for the direct analysis of lubricant and oil additives has been evaluated. All selected additives were successfully detected by DART-MS as standards and in an oil matrix. The surface material, DART helium gas temperature and the presence of an oil matrix were all shown to effect the desorption and ionisation of target analytes. The quantitative capabilities of DART-MS were assessed using the antioxidant additive in a lubricant oil matrix and in the presence of an internal standard. The technique showed good linearity and repeatability. The untargeted analysis of chemical additives present in a fully formulated lubricant oil has been carried out by DESI and DART ionisation techniques. The effect of DESI electrospray solvent and DART helium temperature were both shown to impact the observed mass spectral response for the sample. The analysis of crude oil is particularly problematic due to the high complexity of the sample. A crude oil sample has been analysed using ESI combined with high resolution MS, ESI-FAIMS-MS and DESI-MS. High resolution mass spectrometry enabled the identification of molecular ions that could be characterised using specialist software. The use of FAIMS resulted in shift in the observed chemical profile for the crude oil sample showing selective transmission of molecular species based upon the differential mobility of ions rather than factors such as polarity or solubility that are typically used for sample fractionation. Molecular species from within the crude oil sample were successfully desorbed and ionised by DESI-MS using a DESI solvent composition of 6:4 toluene:methanol.

The presence of harmful algal blooms (HAB) is a growing concern in aquatic environments. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water. Although several analytical approaches have been applied to monitoring cyanobacteria toxins, conventional methods are costly and time-consuming so that analyses take weeks for field sampling and subsequent lab analysis. Capillary electrophoresis (CE) becomes a particularly suitable analytical separation method that can couple very small samples and rapid separations to a wide range of selective and sensitive detection techniques. This paper demonstrates a method for rapid separation and identification of four microcystin variants commonly found in aquatic environments. CE coupled to UV and electrospray ionization time-of-flight mass spectrometry (ESI-TOF) procedures were developed. All four analytes were separated within 6 minutes. The ESI-TOF experiment provides accurate molecular information, which further identifies analytes.

Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis. / Science, Faculty of / Chemistry, Department of / Graduate

The purpose of this thesis is twofold. A special type of signal is studied and methods for its reduction investigated. The results of this work are then applied to the automatic reduction of the trimethyllead mass spectrum with the aim of improving the information recovery from this signal, using modern data processing techniques. The precision measurement of amplitudes must always provide some form of averaging or filtering. It is demonstrated that the results become increasingly sensitive to irregularities in the abscissa of the record, as the width of the averaging function increases. The continuity of the transition from a purely height sensitive calculation to an area sensitive calculation depends on the shape of the impulse response of the measuring arrangement. For all physical shapes, a least squares fit will lead to area sensitive amplitudes. These results have not been known in mass spectrometry or, to our knowledge, in other fields. The mass spectrum is regarded as the convolution of an ideal line spectrum and a peak shape which is determined by the characteristics of the mass spectrometer and the associated electronics. The reduction presents essentially a problem in deconvolution. Although occassionally suggested in the literature, methods involving transformation to the frequency domain were not found to be useful. Thus it is necessary to carry out the analyses entirely in the time domain. Even then, methods requiring the use of derivatives of the spectrum, which have been successfully applied in special cases, usually suffer seriously from noise. Correlation techniques are especially valuable for signals with a high noise content. Correlation is an integration in the time domain and corresponds to a general filtering process in the frequency domain. A close relationship between the correlation technique and a least squares method is emphasized. The development of a practical procedure for this laboratory was part of the study. The trimethyllead group of the tetramethyllead spectrum is recorded in digital form on paper tape. At a constant sampling rate of two per second, about fifty readings per mass interval are recorded. The reduction of the paper tape record to relative isotopic abundances of lead has been automated, applying the experience gained from the general study. The saving in reduction time over former standard methods is substantial. There seems to be a real reduction in the standard deviations of individual peak measurements, but this does not exceed a factor of two. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

The tests of performance of the modified thin-lens magnetic spectrometer using ring-detection have been extended using improved mechanical controls of detector position. The results show only a small improvement over the performance obtained previously in this laboratory. We conclude that the limit of performance with the thin-lens magnet has been reached. Further improvement: may be achieved only with a precision-wound magnet coil which will produce a completely symmetric field. The decay of [formula omitted] has been investigated using the modified thin-lens spectrometer, a scintillation spectrometer and beta-gamma, conversion-electron gamma coincidence techniques. The results support the simpler decay scheme proposed by Van Wijngaarden and Connor. The beta decay has three components with end-point energies and intensities of 659+3 kev(67 .3%), 411 kev (2.5%) and 89 kev (30.3%), estimated from the energy level intensity balances in Ba¹³⁴. These intensity balances show discrepancies of less than 3% of the total decay intensity. The conversion coefficients, calculated from the conversion electron and gamma-ray intensities lead to the following multipolarity identifications for the transitions in Ba¹³⁴; 473 kev (Ml or E2)j 563-569 kev (Ml or E2), 605 kev (E2), 797-803 kev (E2), 1036 kev (Ml or E2), 1168 kev (E2) and 1366 kev (E2), in agreement with other work. The Ml or E2 character of the 473 kev and 1036 kev transitions makes it possible to assign a spin of 3+ or 4+ to the 1641 kev level which was uncertain before. An unsuccessful search for evidence of a 960 kev gamma-ray reported by others puts an upper limit of 0.2% on its intensity. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The first measurements of the radioactive 135Cs and 134Cs isotopes were made on an accelerator mass spectrometer. The natural Ba interference was suppressed using an isobar separator for anions (ISA) in order to measure the less abundant isobaric 134Cs and 135Cs isotopes. It was found that the Ba interference could be suppressed by a factor of 2 × 10^5 while 25% of Cs was transmitted. Furthermore, through comparing the known natural abundance of Ba isotopes to the measured concentration in a sample it was shown that the ISA does not introduce significant mass dependant fractionation at the level of 0.8%. A slow sequential injection analysis technique was developed to measure 135Cs using 134Cs as a reference isotope. This technique also permitted the monitoring of Ba interference. The ionization efficiency of Cs when analyzed in the molecular anion form, CsF2^- , was on the order of 10^−7 while the total measurement efficiency was 1.7×10^−9. The abundance sensitivity of this system was found to be 135Cs/ 133Cs = (1.3 ± 1.7) × 10^−10 , corresponding to a 3σ detection limit of 132.5 pg of analyte per target. Lastly, using the developed AMS techniques, beta spectroscopy, gamma spectroscopy, and isotope production, a measurement of the half life of 135Cs was made. The two measurements of the half life of 135Cs were 0.72 ± 0.32 Ma and 0.99 ± 0.42 Ma.

Bioanalysis is a subdivision of analytical chemistry and deals with biological analytes such as metabolites, proteins, nucleic acids, small molecules, virus particles and entire cells. The rationale of my thesis was to achieve two goals: (i) develop a set of ready to use methods (ii) which are capable providing exact concentrations of analytes as well as kinetic and thermodynamic parameters of their interactions. To investigate interactions between biomolecules special conditions are required which do not interfere with the course if biomolecule interactions. Establishing these conditions and optimization of separation and detection parameters can be tedious and can take longer than actual analysis of samples. I developed a variety of Capillary Electrophoresis – Mass Spectrometry (CE-MS) methods suitable for bionalalysis. CE-MS establishes a new paradigm that separation methods together with MS detection can be used as comprehensive kinetic tools. Most previous attempts to use chromatography and electrophoresis for studying nucleic acid interactions were restricted to assuming slow or no equilibrium between reactants. Kinetic CE (KCE) shows that non-zero kinetics and structural dynamics must be taken into account when separation happens. KCE-MS could be a valuable supplement to IM-MS due to the separation of ions in solution according to their size-to-charge ratio. These methods allowed to reveal new facts about biomolecules and added novel data to the bank of the mankind knowledge. For the best of my knowledge, kinetic parameters for TG2 and thrombin G-quadruplex folding were reported for the first time. I developed a homogeneous method to determine kon, koff and Kd of fast and weak noncovalent interactions between multiple unlabeled ligands (small molecule drugs) and an oligosaccharide (α- or β-cyclodextrin) simultaneously in one capillary microreactor. It has been shown for the first time that KCE can be used to separate and detect the slowly interconverting open and closed conformations of human TG2. It allowed the first direct measurement of the Kd value for calcium binding. Sixteen new substrates were discovered for three aminotransferases (AAT, BCAT, and DAAT). In addition, Viral qCE showed a feasibility to analyse both the count of intact viral particles and sample nucleic acid contamination.

An analog data acquisition system incorporating an Interdata Model 4 digital computer has been designed and built for a mass spectrometer. This system has been conceived with the primary objectives of improving analytical precision and production. Automated mass spectrometer operation allows for the collection of larger quantities of data while decreasing operator involvement and consequently diminishing operator bias and fatigue. The analog signal from the mass spectrometer measuring system is digitized using a digital voltmeter and transmitted, via an interface, to the processor where the digital information is manipulated in accordance with a computer program. An additional facility is provided whereby digital data from the processor can be displayed, if desired, on a 5 decade numerical readout situated at the mass spectrometer console. Hardware is also available in the interface to provide control of the magnetic field scan rate. A function control switch at the mass spectrometer console allows the operator to convey a variety of predetermined instructions to the computer at any time during the course of a run. Thus, the system provides for on-line (real time) data processing of mass spectra as well as limited computer control over the mass spectrometer. This thesis is primarily concerned with the design and construction of the logic hardware for this system together with a demonstration of its operating ability. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subsequently pinpointing the phosphorylation moiety is not straightforward and remains a challenging task. Furthermore, it has been suggested that not all phosphorylation sites are of equal functional importance, to the extent that some may even lack function altogether. Clearly, such sites will confound the efforts towards functional characterisation. The work in this thesis is aimed at these two issues; accurate site localisation and functional annotation. To address the first issue, I adopt a multi-tool approach for identification and site localisation; utilising the different underlying algorithms of each tool and thereby allowing an orthogonal perspective on the same tandem mass spectra. Doing so enhanced accuracy over any single tool by itself. The power of this multi-tool approach stemmed from its ability to not predict more true positives but rather by removal of false positives. For the second issue, I first investigated the hypothesis that those of functional consequence exhibit stronger phosphorylation-characteristic features such as the degree of conservation and disorder. Indeed, it was found that some features were enriched for the functional group. More surprisingly, there were also some that were enriched for the less-functional; suggesting their incorporation into a prediction algorithm would hinder functional prediction. With this in mind, I train and optimise several machine-learning algorithms, using different combinations of features in an attempt to (separately) improve general phosphorylation and functional prediction.