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Optimization of disulfide mapping using mass spectrometryMatsumiya, Nozomi January 1900 (has links)
Master of Science / Biochemistry / John Tomich / One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.
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Investigating the inhibitor and substrate diversity of the JmjC histone demethylasesSchiller, Rachel Shamo January 2016 (has links)
Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N<sup>ε</sup>-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.
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Next-generation Protein Sequencing (NGPS) For Determining Complete Sequences for Unknown Proteins and AntibodiesHoward, Alexis S. 01 January 2021 (has links)
Next-Generation protein sequencing (NGPS) creates newfound ways of fully identifying every protein species in a single biological organism. It is an effort to use technology to determine proteomic data. The purpose of this research project is to use the current technology to sequence proteins and potentially find treatments for some diseases that are common today. Through NGPS, scientists can identify low abundant proteins including those that go through post-translational modifications (PTM) [1]. NGPS will allow us to fully determine protein sequences from protein samples using mass spectrometry with the ultimate goal of being able to determine the primary sequence of the protein in the given sample [1]. Antibodies are a specific class of proteins that aid our bodies in the immune response. Due to their variability in the complementary-determining region (CDR), NGPS will be used to determine the heavy and light chain sequences [2]. The goal of this technology is to fully determine the primary sequence of a protein in a given sample. The randomness of an antibody’s variable (V), diversity (D), and joining (J) genes (VDJ recombination) makes each protein unique. VDJ recombination refers to the process of T cells and B cells randomly assembling different gene segments. This process allows the antibody to make specific receptors that can recognize different molecules presented on the surface of antigens. Proteases are enzymes that break down proteins and peptides. By using different proteases with varying cutting rules, we can digest the antibody and run it through high mass accuracy determining instrument [1]. NGPS allows us to utilize mass spectrometry technology to measure proteins or polypeptides. Because of these measurements, post-translation modifications, including glycosylation, can be detected, unlike in DNA sequencing technology. Protein sequencing has the opportunity to play a major role in the fight against the COVID-19 outbreak and serve as curative measures for the treatment and Type 2 Diabetes [3]. Proteomics can serve as the basis of vaccine development as well as monitoring treatment. Utilizing techniques such as mass spectrometry could reveal the structure of the virus and ultimately allow for engineered tissues to produce the protein in large amounts in a lab setting. Currently, many companies are utilizing highly sensitized technology to carry out the goals of NGPS. The Oxford Nanopore is a company that uses technology to develop and explore more ways to undergo protein analysis. The methods used by this company involve using protein nanopores to mutate residues in pores to determine the overall sequence. The company utilizes modified aptamers that are drawn to the nanopore current. These aptamers can bind with some, but not all pores, allowing for the differentiation between target and non-target proteins. Nicoya Life Sciences is another company that uses Open Surface Plasmon Resonance (SPR) to detect molecular interactions. SPR uses an analyte (a mobile molecule) to bind to a ligand and observe changes in the refractive index. SPR allows researchers the ability to characterize the binding kinetics and affinities of monoclonal antibodies. SPR is an extremely promising technique to sequence proteins due to its flexibility in being able to work with a variety of molecules including lipids, nucleic acids, cells, viruses, nanoparticles, proteins, antibodies, carbohydrates, and more. The original goal behind NGPS was to establish a method to sequence proteins to aid in the early detection of common diseases such as Type 2 Diabetes. After significant research, it is now known that NGPS can be done in a variety of ways to accomplish a common goal—sequencing proteins and understanding how amino acids affect the human body. In the case of diseased states, NGPS can help researchers find ways to diagnose, treat, and cure diseases early on. Focusing on antibodies allows us to manipulate the body’s immune response to rid the host of pathogens. NGPS, however, is advancing at a much slower rate than anticipated by companies due to its many limitations including not being able to sequence large peptides, difficulties in material and composition of the sample, and needing to label small peptides to begin degradation. Ideally, finding a way to combine the high accuracy and specificity of certain techniques, the ability to detect low abundant proteins in others, and the flexibility of Open SPR would allow researchers and companies to create the standard for NGPS. Creating effective antibodies is precisely why NGPS has such great potential today. Ultimately, I found that as a standalone, Open SPR is the most effective method. However, as the research shows, there are limitations with each method, including Open SPR. The conclusion shows that it is necessary to find a combination of these techniques and create an accurate method, potentially using different technologies, to establish the most effective way to sequence proteins.
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Nouvelle méthode en protéomique pour améliorer l'identification et la quantification des protéines acétylées / Developement of a new proteomic method to improve identification and quantification of acetylated proteinsDiallo, Issa 09 November 2017 (has links)
L'acétylation des protéines constitue l’une des plus importantes modifications post-traductionnelles (PTMs). Elle intervient dans de multiples processus bologiques et physiopathologiques tels que, l’activité transcriptionnelle, l'apoptose, la régulation des voies métaboliques, les cancers, les maladies inflammatoires et cardiovasculaires. Face à l’importance de l’acétylation des protéines, il apparaît donc indispensable de bien comprendre les mécanismes qui y sont associés, et donc, de pouvoir identifier et quantifier les protéines acétylées à partir du protéome complet d’échantillons complexes tels que des extraits cellulaires ou tissulaires. La spectrométrie de masse est une technique de choix pour de telles études, car elle permet d’identifier les protéines et les sites d’acétylation, mais aussi de les quantifier en l’associant à des techniques de quantification (label free, SILAC, iTRAQ/TMT, AQUA). Malheureusement, ces méthodes ne ciblent pas particulièrement les acétylations et requièrent l’utilisation de techniques d’enrichissement ou de fractionnement qui ne sont dédiées qu’à certains types d’acetylation : les N-ter et K-acetylation. Aucun enrichissement n’est disponible pour les O- acétylations et ces méthodes d’enrichissement ne sont pas toujours compatibles avec les techniques de quantification citées ci-dessus. Pour améliorer la détection et la quantification des acétylations, nous proposons la méthode RAQIAT (Relatif Absolute Quantification Isobaric Affinity Tag) qui se résume en trois grandes étapes: i) Le blocage des fonctions amines libres à l'aide de la di-méthylation réductrice, ceci empêchera ces dernières de réagir avec le réactif RAQIAT, ii) La désacétylation des lysines acétylées pour permettre une quantification sélective des acétylations, iii) Le marquage des amines primaires précédemment désacétylées dans l’étape 2 par le réactif RAQIAT pour permettre leurs identifications et quantifications. Ce manuscrit a porté en partie sur les deux premières étapes de la méthode RAQIAT.Dans la première étape, les échantillons de protéines de levure ont été digérés puis di-méthylés et fractionnés par OFFGEL en 24 fractions. Ensuite, chacune de ces 24 fractions OFFGEL a été soumise à un fractionnement nano-RPLC et analysée par MALDI TOF/TOF (4800 MALDI-TOF/TOF, Sciex). En parallèle, la même expérience a été réalisée, cette fois-ci sans di-méthylation. L'analyse des données a été réalisée en utilisant le logiciel Mascot comme moteur de recherche.L’efficacité de la réaction de di-méthylation démontrée, nous avons montré que sans réaliser la di-méthylation réductrice 164 sites acétylés ont pu été identifiés alors que 385 sites acétylés distincts ont été identifiés avec la di-méthylation réductrice. De plus, l'amélioration de la détection de l'acétylation en utilisant la méthode de di-méthylation a été observée pour chacune des différents types acétylations: N-ter, K- et O-acétylation.Dans la deuxième étape, nous avons présenté des résultats préliminaires de déacétylation par la sirtuine 1 en présence du peptide de la p53 (Ac-Arg-His-Lys-Lys-(Ac)-AMC) connu comme étant un substrat de cette enzyme. Nous avons observé la formation d’un peptide non acétylé, suggérant une déacétylation de ce peptide acétylé de p53. Cependant, la formation de cet ion étant très faible et l’ion acétylé étant fortement préservé, nous en avons conclu que l’efficacité de la déacétylation du peptide de p53 n’était pas suffisante pour l’intégrer à la méthode RAQIAT. / Protein acetylation is one of the most widespread post-translational modifications which is involved in many cellular physiologies and pathologies such as cancers. Regarding the important biological effect of protein acetylation and a non-negligible number of proteins bearing this PTM, several methods emerged last decade to investigate such PTM. But the detection of acetylations and their quantification are still limited and enrichment method allowing a better detection of acetylation target mostly one kind of acetylation (K-acetylation). To improve the detection of the three kind of acetylation (N-ter, K, and O-) and their quantification, we propose the RAQIAT method (Relative Absolute Quantification Isobaric Affinity Tag), based on protein digestion followed by 3 steps : i) a protection of free primary amines at N-ter, lysine (i.e. primary amine not bearing PTM) based on a reductive di-methylation strategy ii) a deacetylation of acetylated residues to obtain free primary amine corresponding to peptides previously acetylated iii) a RAQIAT labeling on the free primary amine obtained in the previous step to allow the enrichment of peptides previously acetylated and their quantifications. Herein, we present the investigation of the two first steps of RAQIAT method.In the first step, we evidenced that the reductive di-methylation strategy improved the detection of the three kind of acetylation: N-ter, K- and O- acetylations. Yeast protein samples were digested with trypsin prior di-methylation of resulting peptide mixture. Then, di-methylated peptide mixtures were fractionated by OFFGEL and reverse phase liquid chromatography followed by MALDI-TOF/TOF mass spectrometry analysis. Data analysis was performed by using Mascot as search engines.Our results showed that OFFGEL fractionation is a useful step to increase detection of acetylations. Moreover, we showed that our di-methylation treatment improved significantly detection of acetylation. Indeed, after di-methylation treatment, 385 unique acetylated sites were identified while 164 unique acetylated peptides were detected without di-methylation treatment. The improvement of acetylation detection using our di-methylation strategy is observed for each of acetylations: N-ter, K- and O-acetylations. Thus, this new proteomic method is promising to enhance N-ter, K- and O-acetylation detection.In the second step, we presented preliminary results of deacetylation by sirtuin 1 in the presence of p53 peptide (Ac-Arg-His-Lys-Lys- (Ac) –AMC. However, the low deacetylation efficiency of the p53 peptide observed, conclude that is not suitable to applicate into RAQIAT Method
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