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Salmonella species continue to be commonly associated with cases of food-borne disease in developed countries. In the United States in 2001, the incidence per 100,000 people was highest for salmonellosis (15.1), followed by campylobacteriosis (13.8) and shigellosis (6.4). Enteric pathogens usually contaminate the surface of raw animal products during slaughter and primary processing (scalding, defeathering or dehiding, rinsing, cutting, mixing, and grinding, etc.) and can attach and/or reside in the regular and irregular surfaces of the skin, multiply and, thereafter, contaminate food preparation surfaces, hands and utensils. Trisodium phosphate (TSP) has been approved by the USDA as a sanitizer to reduce surface loads of Salmonella on chicken carcasses. A number of studies had demonstrated that TSP effectively removes surface contamination of carcasses by food-borne pathogens. However, very little scientific evidence is available which identifies the actual mechanisms of TSP antimicrobial activity and the response of food-borne pathogens exposed to TSP. This study examined both the physiological and molecular response of Salmonella enterica serovar Enteritidis to TSP treatment. The role of high pH during TSP treatment on its antimicrobial activity was examined. Adaptation of S. enterica serovar Enteritidis to TSP treatment was also examined by analyzing the proteome of serovar Enteritidis cells using two-dimensional gel electrophoresis and mass spectrometry. The role of high pH on the antimicrobial activity of TSP was examined using comparative studies involving treatment solutions containing different concentrations of TSP, treatment solutions adjusted to the equivalent pH as in each of the TSP treatments and TSP solutions pH adjusted to 7.0. Direct and indirect indices of cell survival, membrane damage, and cellular leakage were also employed to examine specific antimicrobial effects. Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH made with NaOH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or NaOH-alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. These results indicate that TSP is a potent membrane-acting agent, and provide compelling evidence that high pH during TSP treatment was responsible for its antimicrobial activity. Adaptation of S. enterica serovar Enteritidis with a sublethal concentration of TSP resulted in the induction of the alkaline stress response. Alkaline stress response involves induced thermotolerance, resistance to higher concentrations of TSP, high pH and sensitivity to acid. Examination of the proteome of TSP-adapted cells revealed differential expression of a number of proteins but did not include the common heat shock proteins involved in thermotolerance. However, TSP adaptation caused a shift in the membrane fatty acid composition from unsaturated to a higher saturated and cyclic fatty acid. This shift in fatty acid composition increases the melting point of the cytoplasmic membrane so that it remains functional at high temperatures. Biofilm bacteria are more resistant to sanitizers, heat and antimicrobial agents than their planktonic counterparts. Examination of the proteome of TSP-adapted biofilm cell of S. enterica serovar Enteritidis revealed little overlap in the protein profile compared to TSP-adapted planktonic cells. Proteomic examination of planktonic and biofilm cells of S. enterica serovar Enteritidis revealed differential expression of a number of proteins involved in DNA replication, stress survival and transport of newly synthesized proteins. These results clearly indicate that changes in the expression of specific genes are involved in the biofilm mode of growth, which could play a significant role in resistance to antimicrobial agents. The results of the current study provide a better understanding of the mechanisms of antimicrobial action of TSP and also elucidate the response of S. enterica serovar Enteritidis to TSP and high pH adaptation. The study also raises new questions regarding stress tolerance of S. Enteritidis following TSP or alkaline pH adaptation with relevance to food safety.

Salmonella species continue to be commonly associated with cases of food-borne disease in developed countries. In the United States in 2001, the incidence per 100,000 people was highest for salmonellosis (15.1), followed by campylobacteriosis (13.8) and shigellosis (6.4). Enteric pathogens usually contaminate the surface of raw animal products during slaughter and primary processing (scalding, defeathering or dehiding, rinsing, cutting, mixing, and grinding, etc.) and can attach and/or reside in the regular and irregular surfaces of the skin, multiply and, thereafter, contaminate food preparation surfaces, hands and utensils. Trisodium phosphate (TSP) has been approved by the USDA as a sanitizer to reduce surface loads of Salmonella on chicken carcasses. A number of studies had demonstrated that TSP effectively removes surface contamination of carcasses by food-borne pathogens. However, very little scientific evidence is available which identifies the actual mechanisms of TSP antimicrobial activity and the response of food-borne pathogens exposed to TSP. This study examined both the physiological and molecular response of Salmonella enterica serovar Enteritidis to TSP treatment. The role of high pH during TSP treatment on its antimicrobial activity was examined. Adaptation of S. enterica serovar Enteritidis to TSP treatment was also examined by analyzing the proteome of serovar Enteritidis cells using two-dimensional gel electrophoresis and mass spectrometry. The role of high pH on the antimicrobial activity of TSP was examined using comparative studies involving treatment solutions containing different concentrations of TSP, treatment solutions adjusted to the equivalent pH as in each of the TSP treatments and TSP solutions pH adjusted to 7.0. Direct and indirect indices of cell survival, membrane damage, and cellular leakage were also employed to examine specific antimicrobial effects. Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH made with NaOH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or NaOH-alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. These results indicate that TSP is a potent membrane-acting agent, and provide compelling evidence that high pH during TSP treatment was responsible for its antimicrobial activity. Adaptation of S. enterica serovar Enteritidis with a sublethal concentration of TSP resulted in the induction of the alkaline stress response. Alkaline stress response involves induced thermotolerance, resistance to higher concentrations of TSP, high pH and sensitivity to acid. Examination of the proteome of TSP-adapted cells revealed differential expression of a number of proteins but did not include the common heat shock proteins involved in thermotolerance. However, TSP adaptation caused a shift in the membrane fatty acid composition from unsaturated to a higher saturated and cyclic fatty acid. This shift in fatty acid composition increases the melting point of the cytoplasmic membrane so that it remains functional at high temperatures. Biofilm bacteria are more resistant to sanitizers, heat and antimicrobial agents than their planktonic counterparts. Examination of the proteome of TSP-adapted biofilm cell of S. enterica serovar Enteritidis revealed little overlap in the protein profile compared to TSP-adapted planktonic cells. Proteomic examination of planktonic and biofilm cells of S. enterica serovar Enteritidis revealed differential expression of a number of proteins involved in DNA replication, stress survival and transport of newly synthesized proteins. These results clearly indicate that changes in the expression of specific genes are involved in the biofilm mode of growth, which could play a significant role in resistance to antimicrobial agents. The results of the current study provide a better understanding of the mechanisms of antimicrobial action of TSP and also elucidate the response of S. enterica serovar Enteritidis to TSP and high pH adaptation. The study also raises new questions regarding stress tolerance of S. Enteritidis following TSP or alkaline pH adaptation with relevance to food safety.

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Mass spectrometry is a versatile, powerful analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry is key component in expanding the capabilities of mass spectrometry beyond just a molecular weight detector. Another key component is the discovery of electrospray ionization allowing not only liquid samples to be ionized but also generation of multiply charged ions enabling mass spectrometry analysis of large biomolecules. The fragmentation pathway of ion during tandem mass spectrometry is highly dependent on the nature of the ion as well as the form of dissociation technique employed. To-date, no single form of ion or dissociation method can provide all the structure information needed; therefore, it is common to use multiple forms of ions, different charge carrier or modifications, with a variety of other dissociation techniques to generate complimentary information. Practically, it is not always easy to generate the desired form of ion via ionization methods and is one of the limitations. Gas-phase ion/ion reactions provide an easy approach in manipulation of ions, either through changing the ion type or covalent modifications, in the gas-phase with the goal of enhancing the capabilities of mass spectrometers for either molecular weight or structural analysis. In this dissertation, studies of new gas-phase ion/ion chemistry for biomolecules such as carbohydrates and phopho/sulfopeptides were performed, and exploration into mass spectrometry analysis of IgGs is discussed. <br><div> Ion/ion reactions with carbohydrates were investigated with the goal of finding a charge-transfer or covalent modification reaction which can increase the structural information of carbohydrates upon tandem mass spectrometry. No luck was achieved with charge transfer ion/ion reactions which increased the overall fragmentation information in tandem mass spectrometry. Novel gas-phase covalent chemistry was discovered where alkoxides were found to form ester and ethers. It was also discovered the aldehyde functional group at the reducing end of carbohydrates are susceptible to Schiff-base modifications. Schiff-base has been previously reported in peptides and this is the first time it has been discovered for carbohydrates.</div><div> In the next project a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions was developed. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate</div><div> Hyaluronic acid, a linear carbohydrate polymer with repeating units of D-glucuronic acid and N-acetyl D-glucosamine, was found to exhibit unique properties in its electrospray ionization mass spectrum that was never seen before. Electrospray of hyaluronic acid in aqueous solution in the negative polarity presented an incredibly intriguing mass spectrum, which we termed “emerald city” consisting of max charge or max charge-1 anions of hyaluronic acid. This is the first biomolecule observed to have the capability to deprotonate at every acid site that is possible. These set of highly charge anions exhibits unique characteristics upon use as a charge inversion reagent to charged invert multiply protonated proteins. A max of thirty-three protons was transferred when myoglobin 24+ was charge-inverted to a max charge state of 9- in the negative mode. Further research should be conducted to fully understand this phenomenal and its possible utilities.<br></div><div> Lastly, mass spectrometry analysis of monoclonal antibodies was performed. Monoclonal antibodies are 150 kDa sized protein complexes and is a major area of interest for pharmaceutical industry. Mass spectrometry analysis of big proteins is an emerging area for mass spectrometry and is quite different compared to small and medium molecule analysis on the mass spectrometer. Detailed in the last chapter are methods developed for sample cleanup of immunoglobulin G as well as the application of q2 DDC for removal of loosely bound adducts to achieve sharper peaks in the mass spectrum. Studies of protein denaturation was also conducted with methods such as circular dichroism and differential ion mobility also employed. And finally, a photochemical reaction setup was shown to cleave twelve out of sixteen total disulfide bonds in the immunoglobulin G within seconds compared to traditional solution phase reactions which can take hours.<br></div>