The thesis consists of two parts targeting the characterization of chromatographic behavior of linear tryptic and cyclic peptides containing oxidized methionine (Met) in reversed-phased chromatography. The retention order of methionine-containing peptide analogues was observed to be the same in both studies: Met oxide < Met dioxide < Met. For linear tryptic peptides, the magnitude of the retention time shift may vary dramatically: from –9 % to +0.36 % acetonitrile. Particularly, large negative retention time shifts are found mostly associated with methionine being in the hydrophobic face of an amphipathic helix. Contrary to previously reported observations, I demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity, this occurs only when methionine is in the N3 position of the N-capping stabilization motif preceding an amphipathic helix. In the second study, the effect of peak splitting was observed for some Met oxide-containing cyclolinopeptides, which most likely appear due to diastereomerization.

Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.

To examine the response of wheat to different temperatures and photoperiods at the proteomic level, a series of experiments was performed at the University of Saskatchewan, College of Agriculture and Bioresources, Department of Plant Science. Tandem-mass spectrometry (MS/MS) was used for protein identification. The iTRAQ approach was used to generate raw data for protein quantification. The Pro Group protein identification software was used for protein identification and quantification of differentially expressed proteins. Despite the input samples being from a plant,the software reported non-plant proteins. The traditional approach used by scientists to deal with this problem is to use sequence alignment software to find close green-plant homologs of the non-plant proteins from a plant-only database. Such a technique is problematic since homology-based sequence similarity does not generally equate to similarity of mass spectra. In this work a more radical approach was investigated and implemented. A bioinformatics pipeline was designed and implemented to report plant proteins misidentified by the Pro Group software. The approach drew its idea from the fact that MS/MS-based protein identification uses peptide fragments/ions bearing unique m/z values in the mass spectra. From the reported non-plant proteins and associated peptides, putative m/z values of the peptides are generated and then used to find alternate hits from a green plant-only database. The pipeline uses three different heuristics, each generating a list of candidate proteins. The proteins reported consistently across the three reported lists have the highest likelihood to be present in the original sample. To evaluate the performance of the pipeline, three separate experiments were performed. A set of known plant peptides, a combination of known plant and non-plant peptides and a set of known non-plant peptides were used as input to the pipeline. For each experiment a stringency value (threshold value) was set by the user. Better results were observed by specifying a tighter stringency; that is, more plant proteins were reported consistently across the three reported lists. The research presented in this thesis shows that m/z values, consideration of unique peptides and accounting for proteins with shorter sequences can be used to identify proteins. These characteristics can be used to identify proteins when limited information is available, in this case a list of non-plant proteins reported as being present in a plant-derived sample. The information available was limited because the original input data was already processed by the Pro Group software. The approach presented here is an alternative to a wet lab scientist using sequence alignment tools, sequence databases, and homology-based search. The pipeline can be enhanced by adding various other modules. The results presented here could be used as a foundation for a further study.

To examine the response of wheat to different temperatures and photoperiods at the proteomic level, a series of experiments was performed at the University of Saskatchewan, College of Agriculture and Bioresources, Department of Plant Science. Tandem-mass spectrometry (MS/MS) was used for protein identification. The iTRAQ approach was used to generate raw data for protein quantification. The Pro Group protein identification software was used for protein identification and quantification of differentially expressed proteins. Despite the input samples being from a plant,the software reported non-plant proteins. The traditional approach used by scientists to deal with this problem is to use sequence alignment software to find close green-plant homologs of the non-plant proteins from a plant-only database. Such a technique is problematic since homology-based sequence similarity does not generally equate to similarity of mass spectra. In this work a more radical approach was investigated and implemented. A bioinformatics pipeline was designed and implemented to report plant proteins misidentified by the Pro Group software. The approach drew its idea from the fact that MS/MS-based protein identification uses peptide fragments/ions bearing unique m/z values in the mass spectra. From the reported non-plant proteins and associated peptides, putative m/z values of the peptides are generated and then used to find alternate hits from a green plant-only database. The pipeline uses three different heuristics, each generating a list of candidate proteins. The proteins reported consistently across the three reported lists have the highest likelihood to be present in the original sample. To evaluate the performance of the pipeline, three separate experiments were performed. A set of known plant peptides, a combination of known plant and non-plant peptides and a set of known non-plant peptides were used as input to the pipeline. For each experiment a stringency value (threshold value) was set by the user. Better results were observed by specifying a tighter stringency; that is, more plant proteins were reported consistently across the three reported lists. The research presented in this thesis shows that m/z values, consideration of unique peptides and accounting for proteins with shorter sequences can be used to identify proteins. These characteristics can be used to identify proteins when limited information is available, in this case a list of non-plant proteins reported as being present in a plant-derived sample. The information available was limited because the original input data was already processed by the Pro Group software. The approach presented here is an alternative to a wet lab scientist using sequence alignment tools, sequence databases, and homology-based search. The pipeline can be enhanced by adding various other modules. The results presented here could be used as a foundation for a further study.

Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.

Background: Bacteriophage Φ6 is a lipid-enveloped dsRNA bacteriophage. The limitations in our knowledge of how this bacteriophage occurs in the environment are limited by non-selective isolation techniques. Research on finding phages in the environment in the past has employed the Double Agar Layer (DAL) plaque assay using Tryptic Soy Agar (TSA), a non-selective media. The bacterial host for bacteriophage Φ6 is Pseudomonas syringae. In this study, we tested Pseudomonas Agar, a selective media that suppresses the growth of bacteria except Pseudomonas species, in the standard double agar layer plaque assay for Φ6. Methods: DAL plaque assays were performed to determine the sensitivity of both Tryptic Soy Agar (TSA) and Pseudomonas Agar (PA) for determining the titer of pure bacteriophage Φ6 stocks. We used Pseudomonas syringae (HB10Y) as the host, and the plaque formation on both agars was compared. Following the evaluation of PA with pure Φ6 stocks, PA effectiveness for Φ6 isolation from environmental samples was tested in spiked waters obtained from irrigation ponds at an agricultural farm. Results: Comparison of TSA and PA using pure Φ6 cultured in the laboratory and spiked environmental samples showed that PA agar can detect bacteriophage Φ6 as well as the standard DAL assay using TSA. On PA, formation of clear visible plaques comparable to the plaques formed using TSA was observed. Conclusions: Pseudomonas Agar can be used for the isolation of bacteriophage Φ6 in environmental samples. This may enhance the detection of these phages in the environment.

The objective of this study was to determine if acoustic emissions (AE) generated by three strains of Escherichia Coli (5024-parent strain, 8279-mutant strain and 8279-random/unrelated strain) could be used to differentiate each strain during their growth cycle. An acoustic sensor with an operating range of 35 kHz-100 kHz was inserted into the growth vessel and attached to a selected channel to capture AE data. The growth vessel was loaded with 60 ml of tryptic soy broth (TSB) (0.25% fructose) media with alginate (1.1%) or without alginate and inoculated with 1% (108 CFU/ml) of an E. coli strain. The growth vessel was placed in a monitoring chamber and incubated at 32°C for 8-9 h. The AE’s generated by each strain were collected throughout the growth cycle. All strains grown in media with and without alginate generated AE’s within 5 min post inoculation. Strains grown in media without alginate generated stronger (P < 0.0001) absolute energy (ABSE) and higher peak frequencies (PFRQ’s), than in media with alginate. The AE’s generated by strains 5024 and 8237 were stronger and easily distinguished from those generated by strain 8279. Strain 8237 generated 12% stronger ABSE from the 3rd to 8th h and 51% stronger PFRQ intensities than strain 5024 during 0-8 h. However, strain 5024 generated 15% stronger ABSE and 31% higher PFRQ’s during the final hour of growth. Strain 5024 generated the highest PFRQ’s from 5-50 kHz, while strain 8237 generated higher frequencies from 100-500 kHz. Fourteen distinguishable differences (P< 0.05) in generated PFRQ’s, between strains 5024 and 8237, were also observed in every 5 kHz increments from 100-500 kHz. Of these differences, strain 8237 generated higher frequencies within eight of the kHz ranges, while strain 5024 generated higher frequencies within six other kHz ranges. These data suggests that all bacteria may generate different AE’s, thus producing a unique “fingerprint” of sound that will allow for its identification.

The effect of sub-lethal chlorine stress on various strains/serotypes of Salmonella on biofilm formation and thermal resistance was studied. The effect of oxidative stress (induced by 150 ppm of chlorine in TSB) on Salmonella biofilm formation on polystyrene and stainless steel surfaces at three temperatures (4°C, 30°C, and room temperature) in nutrient rich (full strength TSB) and nutrient limited conditions (1/10th TSB) was evaluated. On polystyrene surface, chlorine stressed S. Heidelberg (strain ID 72), S. Newport (strain ID 107) and S. Typhimurium (ATCC 14028) formed stronger (P < 0.05) biofilms at 30°C. On stainless steel, the chlorine stressed S. Heidelberg (ATCC 8326) and S. Enteritidis (ATCC 4931) at room temperature formed stronger (P < 0.05) biofilms as compared to the non-stressed control cells. The thermal resistance of short-term (1h) and long-term (27d) chlorine stressed Salmonella Heidelberg and S. Typhimurium were compared with the non-stressed controls at three different temperatures (55°C, 58°C and 61°C) and two growth phases (logarithmic and stationary). The short-term stressed log phase cells (both serotypes) were found to be more sensitive (P< 0.05) to thermal inactivation in TSB. Upon long-term sub-lethal chlorine exposure, Salmonella developed a rugose morphotype on tryptic soy agar at 37°C. The rugose morphotype provided significant thermal protection (P< 0.05) against heat stress as compared to smooth morphotype. In chicken broth, at 55°C, short-term chlorine stressed stationary phase S. Typhimurium displayed a higher D55 value compared to non-stressed cells. The findings from this research reveal that some Salmonella strains have the potential to form stronger biofilms and exhibit higher thermal tolerance upon exposure to sub-lethal chlorine concentration.

La quantification des anticorps monoclonaux (mAbs) dans le plasma est un pré-requis essentiel pour les études PK/PD. Les méthodes de références pour quantifier actuellement les mAbs sont de type ELISA mais les difficultés rencontrées notamment lorsque l’analyse porte sur des mAbs dont la cible pharmacologique est circulante, suggèrent que la spectrométrie de masse serait une alternative intéressante. Appliquée au bevacizumab, la stratégie développée fait appel à la spectrométrie de masse en tandem utilisée en mode MRM (HPLC-ESI-QqQ) et porte sur l’analyse des peptides spécifiques du bevacizumab obtenus à l’issu d’une protéolyse trypsique. La quantification absolue est réalisée à l’aide d’une droite de calibration obtenue à partir du ratio des aires des peptides du bevacizumab et de l’étalon interne. Afin de proposer une méthodologie de quantification de référence, nous avons définie les points clés du développement pour la transposition à d’autre mAbs et comparé les deux stratégies d’étalonnage interne les plus employées : l’une utilisant une protéine analogue et l’autre un peptide marqué par des isotopes stables (SIL-peptide). A travers ce développement la stratégie proposée présente un caractère universel vis-à-vis des anticorps monoclonaux de type IgG dont le traitement des échantillons repose sur une purification par protéine A suivit d’une concentration par ultrafiltration et dont la quantification fait appel à l’approche d’étalonnage interne SIL-peptide. Validée selon les recommandations de la FDA, notre méthode présente les performances analytiques attendues en termes de sensibilité, répétabilité et spécificité pour être appliquée à des études cliniques. / The quantification in plasma of monoclonal antibodies (mAbs) is an essential prerequisite to any PK/PD preclinical and clinical study. To date, reference techniques used to quantify mAbs, rely on enzyme-linked immunosorbent assay (ELISA) but the difficulties encountered in particular when the analysis focuses on the mAbs whose pharmacological target is circulating, suggest that mass spectrometry would be an interesting alternative. Applied to bevacizumab, the quantification developed strategy involves tandem mass spectrometry (HPLC-ESI-QqQ) used in MRM mode and focuses on the analysis of specific peptides bevacizumab obtained after tryptic proteolysis. Absolute quantification is achieved through calibration curve obtained from peak area ratios of bevacizumab surrogate peptide and internal standard. To propose a reference quantification methodology, we have identified the key points of development for transposition to other mAbs and compared the two most commonly used internal calibration approaches: one using protein analogue and the other a stable isotope labeled surrogate peptide (SIL-peptide). Through this development, the proposed strategy has a universal character with respect to IgG monoclonal antibodies subclasses which is based on sample processing purification using protein A followed by concentration by ultra filtration and whose quantification involves the internal calibration approach SIL-peptide. Validated according to FDA guidelines, our method shows the expected analytical performance in terms of sensitivity, specificity and repeatability for application in clinical studies