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Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

A proteomic procedure for analyzing mouse left ventricular collagen by mass spectrometry has been developed. The procedure involves a purification step that removes non-collagenous cellular components from the collagen extracellular matrix, a step that solubilizes the collagen in aqueous solvents before it is proteolytically digested for analysis with ESI-LCMS/MS. Collagen from healthy and lathrytic mice has been positively identified by applying the SEQUEST database search algorithm to spectra from the collagen prepared using this procedure. Analysis shows that the relative percentage of collagen peptides detected in lathrytic tissue is significantly greater than that of the healthy tissue. These preliminary results suggest that the percentage of cross-linked collagen is lower in the lathrytic tissue as indicated by the greater protein sequence coverage obtained for this tissue. This procedure lays the ground work for future experimentation that has the ability to allow for the identification and quantification of cross-linked peptides.

Burkholderia pseudomallei is the causative agent of Melioidosis, an endemic disease in South East Asia, and is classified as a category B biological agent. Currently, there is no licensed vaccine for this disease; the mortality rate is high due to the incorrect diagnosis and the pathogen insusceptibility to general antibiotics. A mass spectrometry based proteomic approach has been applied in order to identify the proteins that are responsible for pathogenicity.Methods were developed for the proteomic analysis of Burkholderia species using B. vietnamiensis G4, an opportunistic pathogen as the model organism. Both gel-based (LC-MS/MS) and gel-free MudPIT (LC/LC-MS/MS) approaches have been applied for the analysis of the proteins extracted from four different cellular fractions of these bacteria. More than 1200 proteins were identified from these analyses, including many proteins previously identified as virulence factors of these bacteria. Similar methodologies were applied to build a proteome map of non-pathogenic B. thailandensis E264 to use as a reference for the pathogenic studies. Additionally, proteomes of two B. thailandensis strains isolated from two geographical locations were compared to investigate the differences in protein expression of these organisms.Proteins identified from pathogenic B. pseudomallei were compared with the non-pathogenic B. thailandensis and opportunistic pathogen B. vietnamiensis proteins. Many species specific proteins were identified from this proteomic analyses; those proteins can be used as antigen targets to selectively identify these pathogenic bacteria in a complex biological matrix using affinity capture methods.Ticks are vectors that can transmit disease causing pathogens one host to another. Knowing the pathogen reservoir is important in order to control disease spread in the environment. Application of mass spectrometric methods to identify the host blood components from tick vectors was investigated using tick nymphs which feed only once in their life cycle. Using mass spectrometry based proteomics; host specific proteins like hemoglobin and immunoglobulin were identified from a single tick nymph analysis. Additional studies have examined the fatty acid profiles of rabbit and sheep blood fed tick nymphs using SPALDI mass spectrometry. Different fatty acid profiles were obtained for these tick nymphs, but further investigations are required to validate these findings.

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-Ion Mobility-orthogonal-TOF mass spectrometer is demonstrated. The early version of the instrument used for proof-of-concept experiments achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (ca. 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOFMS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (peptide mass mapping) and partial sequence analysis, (real time tandem mass spectrometry.) Optimization of the MALDI-IM-SID-o-TOF mass spectrometer for peptide sequencing is discussed. SID spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. Optimum collision energies differ for the various surfaces. The fragmentation patterns observed for a series of peptides and protein digests using the Nd:YAG laser (355 nm) for MALDI ion formation and an FSAM surface for ion activation is compared to the fragmentation patterns observed for CID and photodissociation. The fragmentation patterns observed in all cases are strikingly similar. Photodissociation produced a greater abundance of ions resulting from side-chain cleavages. As a general rule optimized SID spectra contain fewer immonium ions than either photodissociation or CID. Evaluation of an instrument incorporating a new hybrid drift cell is discussed. Spectra for a digest of hemoglobin is compared to that acquired with an ABI 4700 TOF-TOF. The performance of the instrument is also evaluated using a micro-crystal Nd:YAG laser (355 nm) for MALDI operated at 400 Hz. Experiments were performed to determine the sensitivity and overall performance of the instrument. The reproducibility of the MS/MS spectra for gramicidin S is shown to be 94% run-to-run. The best mobility resolution obtained for a neat deposition of the dye Crystal Violet was 60 t/∆t. Sensitivity was tested with the peptide fibrinopeptide A (m/z 1537, AA sequence ADSGEGDFLAEGGGVR). Data acquired for sixty seconds with approximately sixty femtomoles deposited. Abundant [M+H]+ ions where observed as well as [M+H]+-NH3 ions. The S/N for this short run was insufficient to identify any SID fragments

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High-throughput techniques are currently some of the most promising methods to study molecular biology, with the potential to improve medicine and enable new biological applications. In proteomics, the large scale study of proteins, the leading method is mass spectrometry. At present researchers can routinely identify and quantify thousands of proteins in a single experiment with the technique called shotgun proteomics. A challenge of these experiments is the computational analysis and the interpretation of the mass spectra. A shotgun proteomics experiment easily generates tens of thousands of spectra, each thought to represent a peptide from a protein. Due to the immense biological and technical complexity, however, our computational tools often misinterpret these spectra and derive incorrect peptides. As a consequence, the biological interpretation of the experiment relies heavily on the statistical confidence that we estimate for the identifications. In this thesis, I have included four articles from my research on the accuracy of the statistical confidence estimates in shotgun proteomics, how to accomplish and evaluate it. In the first two papers a new method to use pre-characterized protein samples to evaluate this accuracy is presented. The third paper deals with how to avoid statistical inaccuracies when using machine learning techniques to analyze the data. In the fourth paper, we present a new tool for analyzing shotgun proteomics results, and evaluate the accuracy of  its statistical estimates using the method from the first papers. The work I have included here can facilitate the development of new and accurate computational tools in mass spectrometry-based proteomics. Such tools will help making the interpretation of the spectra and the downstream biological conclusions more reliable.

Clathrin-mediated endocytosis (CME) plays a central role in the regulation of multiple cellular processes such as uptake of nutrients, recycling of housekeeping receptors and transporters, as well as for cell surface removal and downregulation of signaling receptors. Once endocytosed, cargo passes through early endosomes where sorting mechanisms traffic the cargo to the recycling pathway or to degradation in the lysosome. The general objectives of this doctoral research were to identify and characterize new players of the clathrin-mediated trafficking pathway to reveal differences between the abundant components of the trafficking machinery in two tissues, and to examine the mechanisms of endosomal sorting. / We used subcellular proteomics to reveal the differences in components of clathrin-coated vesicles (CCVs) isolated from brain and liver and to identify new molecules participating in clathrin trafficking. We demonstrated that the ratio between the clathrin adaptor proteins AP-1 and AP-2 is different in brain and liver, which indicates differential functions between the two tissues. We also discovered that clathrin-light chains, which have been proposed for many years to be regulatory proteins in the assembly of CCVs, were less abundant relative to clathrin-heavy chain in liver and in non-brain tissues compared to brain. / We identified a new DnaJ domain-containing protein, receptor-mediated endocytosis protein 8 (RME-8) that was detected in liver CCVs specifically. Further characterization revealed that the RME-8 DnaJ domain binds to the chaperone heat-shock cognate 70 (Hsc70) in an ATP-dependent manner. RME-8 is a ubiquitously expressed protein that tightly associates with endosomes, and its depletion causes intracellular trafficking defects. Moreover, we demonstrated that RME-8 depletion also leads to a decrease in levels of epidermal growth factor receptor (EGFR), as a result of an increase in EGFR degradation. RME-8 knock-down causes decreased EGFR levels even in cancer cells lines where EGFR is generally protected from degradation. / Globally this doctoral project revealed new insights on specialized functions for c1athrin-mediated trafficking in different tissues and allowed the identification and characterization of a novel protein implicated in sorting decisions occurring on endosomes.

Platelet products have a short shelf life (5 to 7 days) owing in part to the deterioration of the quality of platelets stored at 22°C. This creates significant inventory challenges, and blood banks may suffer shortages and high wastage as a result. Proteomics offers a global quantitative approach to investigate changes occurring in stored blood products. These data sets can identify processes leading to storage-associated losses of blood component quality such as the platelet storage lesion (PSL). Changes to the platelet proteome between days 1 and 7 of storage were analysed with 3 complementary proteomic approaches with final mass spectrometric (MS) analysis: 2-dimensional (2D) gel electrophoresis/differential gel electrophoresis (DIGE), isobaric tagging for relative and absolute quantification (iTRAQ), and isotope-coded affinity tagging (ICAT). Although proteomics analyses identified many storage-associated protein changes, these varied significantly by method suggesting that a combination of protein-centric (2D gel or DIGE) and peptide-centric (iTRAQ or ICAT) approaches is necessary to acquire the most informative data. Validation of the proteomics results by western blotting, flow cytometry, quantitative real-time polymerase chain reaction (qRT PCR) and ³ٰ⁵S-methionine incorporation confirmed that platelets are capable of synthesising biologically relevant proteins ex vivo throughout a 10-day storage period with particularly long-lived mRNA (half-life of approximately 2.4 days), and has provided the first evidence for one of the mechanisms of the PSL. The development of an ³ٰ⁵Smethionine assay has since shown that stored human blood platelets incorporate ³ٰ⁵S-methionine at a rate that is proportional to time and substrate concentration, and is slower for freshly drawn platelets than those stored in pooled buffy coat derived units for 10 days. More interesting still are the observations that the overall ³ٰ⁵S-methionine incorporation rate was higher in pooled buffy coat platelet units versus freshly drawn platelets, that this rate increased upon agonist exposure in both, and that day 8 platelets showed significantly greater total protein translation than on days 2,3,7 and 10 of storage. This may be indicative of translational regulation of the platelet proteome during storage and upon activation. Translational control is a consequence of remarkable cellular specialisation and precise biochemical pathways which, in the case of platelets, may lead to storage-associated losses of blood component quality and must be understood if platelet storage times are to be extended.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.