A method of solubilizing and extracting proteins from by-products was tested. The raw materials used were finely homogenized and digested at 60(DEGREES)C and pH 10.5 for 30 to 120 minutes in the presence of 0.5% alkaline nonspecific bacterial proteases from Bacillus subtillis. The protein in solution was separated from nonsoluble and organic solvent soluble components by filtration or centrifugation. When desired, the proteinaceous solution was dried (preferably by spray drying). Raw materials that were test digested included keratin from turkey feathers, bovine skin collagen, shark waste, shrimp heads, whole squid, inedible chicken carcass, bovine blood plasma, slaughterhouse waste, cotton gin waste, Enteromorpha sp. (a marine alga), Batis sp. and Distycilus sp. (two halophytes), soybean meal, casein, and fibrinogen. With this method, plant proteins were 57.4% to 59.9% extractable and animal proteins were 75.8% to 93.0% extractable. The native protein hydrolyzed by the procedure was reduced to an average molecular weight of 10,000-15,000 daltons. Other changes characteristic of the digestion process were increased protein concentration and decreased ash concentration. Complementation of by-product proteins in Tetrahymena medium resulted in increased growth compared to Tetrahymena cultures using soy or casein as the sole protein source up to 1.25 times. Decreasing protein molecular weight resulted in decreased growth in Tetrahymena (up to 4 times). Shrimp fed hydrolyzed animal proteins grew only 37.6% to 54.8% as much as squid-fed shrimp controls. White leghorn chicks fed 40% protein as hydrolyzed by-product proteins grew as much as chicks fed a commercial-type milo-soy diet supplemented with methionine. Amino acids from smaller peptides were more rapidly absorbed and more completely incorporated into muscle mass by chicks than were larger peptides.

The concentrated protein fractions of soyabeans, known as soya protein isolate, was produced by three different methods from the same raw material namely defatted soya flakes. Extraction of the soluble fraction of the raw material is common to all three methods. A study was therefore undertaken to optimise the extraction process conditions in terms of time, temperature, pH, extraction time, extraction volume and raw material particle size, thereby maximising yields of soluble material. The three different methods, namely isoelectric precipitation, ultrafiltration and swollen gel technology were then used to separate the soluble and non-soluble protein fractions. Both the isoelectric and ultrafiltration methods gave good yields of finished product, with the ultrafiltration process giving the better overall yield, but the swollen gel method gave disappointing results and was not feasible in practice. Functional properties of the products from the isoelectric and ultrafiltration methods were compared and found to be broadly similar although different in certain respects from those of commercial soya isolates. Levels of the anti-nutritional factors trypsin inhibitor and phytate in products from the three processes were determined and the substantial differences observed in trypsin inhibitor levels were further investigated. Determination of lysinoalanine levels was also attempted but the results obtained were unsatisfactory. Amino acid composition and polyacrylamide gel electrophoresis were used to compare the chemical composition of products from the three processes. The comparative economics of the isoelectric and ultrafiltration processes for large scale production of soya protein isolates were evaluated, taking into account the comparative efficiencies of the two processes as determined during the study. It was established that, while the isoelectric process initially appears more economical, it may be possible to modify the ultrafiltration process in such a manner as to make it more economical than the isoelectric process. Overall figures however indicate that the manufacture of soya protein isolate in South Africa is not currently a viable economic proposition, due to high raw material costs. / Thesis (M.Sc.)-University of Natal, 1995.

Chen, Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 75-78). / Abstracts in English and Chinese. / Isolation and Identification of Differentially Expressed Protein in Serum of Patients with Sleep Disorders --- p.I / Abstract --- p.IV / 論文摘要 --- p.VII / Acknowledgements --- p.IX / Table of Contents --- p.X / List of Figures --- p.XII / List of Tables --- p.XII / List of Abbreviations --- p.XIII / Chapter Chapter 1: --- Introduction --- p.2 / Chapter 1.1 --- Definition of narcolepsy --- p.2 / Chapter 1.2 --- Symptoms of narcolepsy --- p.2 / Chapter 1.2.1 --- Excessive Daytime Sleepiness (EDS) --- p.2 / Chapter 1.2.2 --- Cataplexy --- p.2 / Chapter 1.2.3 --- Associated features --- p.3 / Chapter 1.3 --- Prevalence of narcolepsy --- p.4 / Chapter 1.4 --- Pathophysiology and molecular genetics of narcolepsy --- p.7 / Chapter 1.4.1 --- Pathophysiology of narcolepsy --- p.7 / Chapter 1.4.2 --- Molecular genetics research --- p.8 / Chapter 1.5 --- Diagnostic criteria for narcolepsy --- p.12 / Chapter 1.6 --- Treatment of narcolepsy --- p.16 / Chapter 1.7 --- The Burden of narcolepsy --- p.18 / Chapter 1.8 --- Human blood serum/plasma --- p.19 / Chapter 1.9 --- Cerebrospinal fluid (CSF) --- p.23 / Chapter 1.10 --- Aims of study --- p.26 / Chapter Chapter 2: --- Materials and Methods --- p.28 / Chapter 2.1 --- Participants and measurements --- p.28 / Chapter 2.1.1 --- Participants --- p.28 / Chapter 2.1.2 --- Diagnosis measurements --- p.28 / Chapter 2.2 --- "Serum extraction, albumin and IgG depletion" --- p.30 / Chapter 2.2.1 --- Albumin and IgG Depletion Kit --- p.30 / Chapter 2.2.2 --- Chemicals and reagents --- p.30 / Chapter 2.2.3 --- Preparation of solutions --- p.30 / Chapter 2.2.4 --- Procedure --- p.30 / Chapter 2.3 --- Reversed Phase High Performance Liquid Chromatography (RP-HPLC) --- p.32 / Chapter 2.3.1 --- RP-HPLC method --- p.32 / Chapter 2.3.2 --- Chemicals and reagents --- p.33 / Chapter 2.3.3 --- Preparation of mobile phases --- p.33 / Chapter 2.3.4 --- Procedure --- p.33 / Chapter 2.4 --- MALDI-TOF/TOF Mass Spectrometry --- p.35 / Chapter 2.4.1 --- Chemicals and reagents --- p.35 / Chapter 2.4.2 --- Preparation of solutions --- p.35 / Chapter 2.4.3 --- Procedure --- p.35 / Chapter 2.5 --- SDS-PAGE and double staining --- p.37 / Chapter 2.5.1 --- Chemicals and reagents --- p.37 / Chapter 2.5.2 --- Preparation of solutions --- p.37 / Chapter 2.5.3 --- Procedure --- p.39 / Chapter 2.6 --- N-terminal amino acid analysis --- p.42 / Chapter 2.6.1 --- Procedure --- p.42 / Chapter 2.6.2 --- Sequence analysis --- p.42 / Chapter 2.7 --- CSF analysis --- p.43 / Chapter Chapter 3: --- Results --- p.45 / Chapter 3.1 --- Albumin and IgG depletion of human serum samples --- p.45 / Chapter 3.2 --- Peak identification --- p.47 / Chapter 3.2.1 --- Peak identification on HPLC profiles --- p.47 / Chapter 3.2.2 --- Statistical results --- p.51 / Chapter 3.2.3 --- Family cases analysis --- p.54 / Chapter 3.3 --- MALDI-TOF/TOF Mass Spectrometry --- p.56 / Chapter 3.4 --- SDS-PAGE and double staining --- p.58 / Chapter 3.5 --- Protein sequence analysis --- p.60 / Chapter 3.6 --- Cerebrospinal fluid (CSF) analysis --- p.62 / Chapter Chapter 4: --- Discussion --- p.65 / Chapter 4.1 --- RP-HPLC methods --- p.65 / Chapter 4.2 --- The detected peptide fragment and Hlark --- p.66 / Chapter 4.2.1 --- "Human Lark protein (Hlark, hlark)" --- p.66 / Chapter 4.2.2 --- Circadian clocks --- p.67 / Chapter 4.2.3 --- "Hlark, circadian rhythm and narcolepsy" --- p.71 / Chapter 4.3 --- Familial and genetic analysis --- p.72 / Chapter 4.4 --- Clinical implications --- p.73 / Chapter 4.5 --- Conclusion --- p.74 / References --- p.75

Orientador: Elias Basile Tambourgi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-28T22:32:01Z (GMT). No. of bitstreams: 1 Rodrigues_ElianaMariaGoncalves_D.pdf: 3569293 bytes, checksum: b7c2f26dc727eabf16b3528d0a1813bb (MD5) Previous issue date: 2001 / Resumo: Neste trabalho, foi estudada uma microcoluna agitada por campânulas pulsadas, visando promover um eficiente contato entre as fases através de uma agitação suave, aumentando assim o tempo de contato entre elas no interior da microcoluna e também evitando a desnaturação da enzima. O objetivo deste estudo visou a recuperação da xilanase, produzida pelo fungo Penicillium janthinellum, através da técnica de extração líquido - líquido por micela reversa, para o interior micelar. Para tanto, foi utilizado o agente tensoativo catiônico BDBAC (c1oreto de benzil dodecil bis (hidroxietil) amônio). Foram utilizados planejamentos estatísticos com o intuito de se realizar uma triagem das variáveis significativas no processo: freqüência de pulsação das campânulas, razão entre as fases aquosa/orgânica e condutividade da fase aquosa; a metodologia de superficie de resposta foi empregada para a quantificação dos níveis das mesmas. O trabalho resultou em dois modelos matemáticos, um que representa a recuperação da enzima pura, e outro que representa a recuperação da enzima presente no extrato enzimático bruto. Foram previstos e observados experimentalmente para ambos os modelos obtidos, rendimento em atividade na ordem de 140% para a enzima pura e 43% para o extrato enzimático. Verificou-se neste trabalho que, a metodologia estatística empregada foi de extrema importância e que, a microcoluna estudada tem operação estável e altos rendimentos em atividade foram alcançados / Abstract: In this work, it was studied a microcolumn agitated by puIsed caps, due to promote an efficient contact between the phases in the column and also to avoid the enzyme denaturation and the loss of main proteins properties. This work deals with the purification of xylanase produced by Penicillium janthinellum by liquid-liquid extraction on reverse micelles, to micellar inner, utilizing a cationic surfactant BDBAC (N-benzyI-N-dodecyl-N-bis(2-hydroxyethyl)). It was used design statistical with the intention of realized selection of the main variables in the process: frequency pulsed, volumetric flow and ionic strength; the response surface methodology was employed to the quantified levels of this. This resulted in two major mathematical models: one representing the use of pure enzyme and the other the use of enzymatic extract. It was predict and observed experimentally for both models, obtained activity yield in the order of 140% to the pure enzyme and 43% to the enzymatic extract. It was observed in this work that, the methodology statistical employed was extremely important and that the microcolumn used were stable operation and that high activity yield was reached. / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Orientador: Sônia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T04:00:44Z (GMT). No. of bitstreams: 1 Oliveira_CarlaReis_M.pdf: 2012737 bytes, checksum: 29446d9a4e9ff14301c100dcd3fb82ab (MD5) Previous issue date: 2014 / Resumo: Anticorpos monoclonais (AcM) são imunoglobulinas secretadas por clones de linfócitos B imortalizados obtidos pela tecnologia de hibridomas. Esses clones são cultivados em meios de cultura complexos e, geralmente, liberam baixas concentrações de AcM, dificultando as etapas de purificação. Como a utilização destas proteínas nas áreas terapêutica e analítica requer um alto grau de pureza, diferentes estratégias de purificação vêm sendo desenvolvidas, mas usualmente os anticorpos são purificados por cromatografia de afinidade com proteína A ou G imobilizada, o que representa um elevado custo de produção para processos industriais. No intuito de contribuir para o desenvolvimento de processos de purificação dessas moléculas, estudou-se a purificação do AcM anti-Trypanosoma cruzi isotipo IgG2a em orto-fosfo-serina (OPS) imobilizado em gel de agarose. Contrário ao relatado em literatura, observou-se que o agente quelante apresentou baixa capacidade em imobilizar o íon metalico Ni2+. Além disso, o quelato formado adsorveu a molécula alvo juntamente com impurezas do sobrenadante do meio de cultura. Foi observado também que o OPS se comporta como ligante de troca iônica seletivo para IgG2a em solução tampão Tris-HCl 50 mmol/L pH 7,0, quando imobilizado em gel de agarose ativado com CNBr, porém os ensaios realizados com o ligante imobilizado em gel ativado com bisoxirano demonstraram que a inserção de uma molécula espaçadora reduz a seletividade do adsorvente. O AcM foi recuperado com elevado grau de pureza quando empregado em solução tampão Tris-HCl 50 mmol/L pH 7,0 e dessorção pelo acréscimo de NaCl 1,0 mol/L para promover aumento da força iônica. Os resultados de ensaios dinâmicos revelaram que a eficiência de recuperação do produto foi de 97,3% e a capacidade dinâmica de adsorção foi 0,39 mg de AcM/mL de gel devido. Este trabalho sugere a potencialidade de utilização do ligante OPS para a purificação dos anticorpos anti-Trypanosoma cruzi (IgG2a) / Abstract: Monoclonal antibodies (mAb) are immunoglobulins produced by lynphocyte B clones immortalized by the hibridoma technology. Those clones are cultivated in complex medium and generally produce low levels of mAb, interfering on purificaton steps. Since these antibodies are required for analytical and therapeutical purposes, the need of highly pure antibodies is imperative. Although they are usually purified by chromatografic techniques utilizing immobilized protein A ligands, different strategies for downstream processes have been studied to overcome the high costs of these conventional medias for industrial scale purposes. Aiming to contribute with the development of such processes, the anti-Trypanosoma cruzi mAb (IgG2a isotype) was investigated under chromatographic conditons twards the affinity ligand ortho-phospho-L-serine (OPS) immobilized onto agarose beads. It was observed that despite the fact this ligand has been reported as a chelating agent for the purification of immunoglobulins by IMAC technique, OPS presented low chelating capacity for Ni2+ and the metal complex formed adsorbed the desired protein within culture medium contaminants. It was also observed that under buffering conditions of Tris-HCl 50 mmol/L pH 7.0 OPS behaves as a selective ionic exchanger ligand for IgG2a when immobilized in CNBr activated agarose. On the other hand, when immobilized in bisoxirane activated agarose, OPS has shown that the presence of a spacer arm reduces the selectivity of the adsorbent. The mAb was recuperated in one single step with highly putity degree when operated with adsorption buffer Tris-HCl 50 mmol/L pH 7.0 and elution by increasing the ionic strength with the addition of NaCl 1.0 mol/L. The dynamic tests results showed that the efficiency for product recovery was 97.3% and the dynamic binding capacity was 0.39 mg of mAb/mL swollen agarose. This work suggests the potencial use os OPS ligand affinity for the purification of IgG2a antibodies anti-Trypanosoma cruzi in a single step / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T20:44:35Z (GMT). No. of bitstreams: 1 Serracchiani_MarcelMafei_M.pdf: 2065791 bytes, checksum: 04a95a39ac0691a669c45efc9e3fc435 (MD5) Previous issue date: 2013 / Resumo: A produção de proteínas recombinantes em plantas transgênicas tem se mostrado uma forma segura, eficiente e barata de obtenção em grande escala de várias proteínas de interesse, tais como vacinas, enzimas industriais, bioativos, biofarmacos e anticorpos e seus fragmentos. Contudo, para que a produção de proteínas recombinantes em plantas se torne viável é necessário o desenvolvimento de um processo de recuperação e separação da molécula alvo que seja eficiente e reprodutivo, pois a produção de biomoléculas em plantas transgênicas, assim como os métodos tradicionais de produção, apresentam componentes indesejáveis que devem ser removidos. Neste trabalho, avaliou-se a purificação de fragmentos Fab de IgG humana adicionados artificialmente (spiking) a extrato protéico de soja não transgênica utilizando-se a técnica de cromatografia de afinidade por íons metálicos imobilizados (IMAC). Estudou-se o efeito dos quelatos IDA-Ni(II) e TREN-Ni(II), dos sistemas tamponantes Tris-HCl, fosfato de sódio e Mes, na presença e na ausência de sal (NaCl) na purificação de fragmentos Fab. Primeiramente foram realizados experimentos cromatográficos com as proteínas nativas do extrato proteico do grão de soja. Dos resultados obtidos, selecionou-se, para o adsorvente agarose-TREN-Ni(II), o sistema tamponante Tris-HCl/Tris-HCl na ausência de NaCl, e para o adsorvente agarose-IDA-Ni(II), selecionou-se o sistema tamponante fosfato de sódio/acetato de sódio contendo NaCl. Estes sistemas tamponantes e adsorventes foram utilizados para purificação dos fragmentos Fab adicionados (spiking) ao extrato proteico de grãos de soja. Em agarose-TREN-Ni(II), os fragmentos Fab foram purificados por cromatografia negativa, obtendo-se 66% dos fragmentos Fab na etapa de flowthrough e lavagem, com um grau de pureza de 91% e fator de purificação de 1,4. Para o adsorvente agarose-IDA-Ni(II), os fragmentos Fab foram purificados por cromatografia tradicional (eluição a pH 5,8), obtendo-se alto grau de pureza do fragmento Fab purificados, com um fator de purificação de 2,5. Este estudo contribuiu para obter conhecimento de base para posterior aplicação da técnica de IMAC na purificação de proteínas recombinantes produzidas em sementes de plantas transgênicas / Abstract: The recombinant proteins production using transgenic plants have been considered a safe, efficient and cheap way of producing on large scale innumerous proteins of interest, such as vaccines, industrial enzymes, bioactives, biopharmaceuticals and antibody and it fragments. However, for the production of recombinant proteins in plants become viable is necessary to develop a process for recovery and separation of the target molecule that is efficient and reproductive, because the production of biomolecules in transgenic plants, as well as traditional production methods, have components reactions which must be removed. In this work, the purification of Fab fragments of human IgG added artificially (spiking) to extract non-transgenic soy protein, using the technique of affinity chromatography on immobilized metal ions (IMAC), was evaluated. The effect of chelating agents IDA-Ni(II) and TREN-Ni(II), and the buffers Tris, sodium phosphate and Mes, in presence and the absent of NaCl, was studied for the Fab purification. First, the chromatographic experiments with the native proteins of soybean extract were performed. From the results obtained, it was selected, for the adsorbent TREN-Ni(II), the buffers system Tris-HCl/Tris-HCl without NaCl, and for the adsorbent IDA-Ni(II), the buffer system sodium phosphate/ sodium acetate with NaCl was selected. These adsorbents and buffer systems were used for purification of Fab fragments added (spiking) to soybean extract proteins. In the agarose adsorbent TREN-Ni (II), Fab fragments were purified by negative chromatography, obtaining 66% of Fab fragments in flowthrough and wash steps with a purity of 91% and purification factor of 1.4. For the adsorbent agarose-IDA-Ni (II), Fab fragments were purified by traditional chromatography (elution at pH 5.8), obtaining a high purity of the purified FAB, with a purification factor of 2.5. This study contributed to obtain knowledge base for application of the technique on the IMAC purification of recombinant proteins produced in transgenic plant seeds / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Everson Alves Miranda / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T23:00:08Z (GMT). No. of bitstreams: 1 Lima_LeonardoHenriqueFrancade_M.pdf: 992316 bytes, checksum: 57f2f7216633dfbabec7d916c3adb88d (MD5) Previous issue date: 2006 / Resumo: A precipitação de proteínas através da adição de sais (por exemplo, o sulfato de amônio e cloreto de sódio) é uma técnica comumente utilizada em recuperação e purificação de proteínas. Contudo, a remoção de sais do precipitado e tratamento da solução remanescente contendo altas concentrações de sal são etapas limitantes, devido ao custo do processo e a regulamentação ambiental. O uso de eletrólitos voláteis é uma alternativa aos sais convencionais neste tipo de processo, visto permitirem um fácil processamento e redução do custo do tratamento de efluentes: os sais voláteis podem ser removidos com a redução de pressão ou elevação da temperatura. Neste trabalho foram determinadas as curvas de solubilidade para a lisozima e insulinas bovina e suína em soluções aquosas de sais voláteis, em sistema vedado contendo carbonato, carbamato e bicarbonato de amônio em equilíbrio com CO2 e NH3. Estas curvas de equilíbrio foram determinadas como função da concentração salina (1,00 a 7,00 mol.kg-1), da temperatura (5,0 a 25,0 °C), e razão nitrogênio por carbono (RN/C) (2,0 e 2,5) das soluções.O aumento do conteúdo de nitrogênio aumentou a solubilidade das proteínas que, de uma forma geral, apresentaram um comportamento de solubilidade retrógrada em relação à temperatura. Espectros de dicroísmo circular sugeriram uma pequena desnaturação causada pela precipitação / Abstract: Protein precipitation induced by salt addition (e.g., ammonium sulfate and sodium chloride) is a commoly used technique in the downstream processing of proteins. However, salt removal from the precipitate and disposal of the salt containing liquid phase are key steps of the process due to cost and environmental concerns. Volatile salts are alternatives to conventional salts in this process since they can allow easy processing and reduce the cost of waste disposal: the volatile salt can be removed by pressure reduction and temperature increase. In this work we reported the solubility curves for lysozyme and swine and bovine insulins in aqueous solutions of the volatile salts of the system comprised of ammonium carbonate, carbamate, and bicarbonate in equilibrium with CO2 and NH3. These equilibrium curves were determined as funciton of the salt concentration (1.0-7.0 mol/kg), temperature (5.0-25.0 ºC), and N/C ratio (2.0 and 2.5) of the solutions. The increase of the N content of the solutions increased the proteins solubility that in general had a retrograde solubility. Circular dichroism spectra suggested some denaturation caused by the procipitation / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)