Дисертації з теми "Crosslinking mass spectrometry"

The 3D structures of proteins may provide important clues to their functions and roles in complex biological pathways. Traditional methods such as X-ray crystallography and NMR are not feasible for all proteins, while theoretical models are typically not validated by experimental data. This project investigates the use of chemical crosslinkers as an experimental means of validating these models. Five target proteins were successfully purified from yeast whole cell extract: Transketolase (TKL1), inorganic pyrophosphatase (IPP1), amidotransferase/cyclase HIS7, phosphoglycerate kinase (PGK1) and enolase (ENO1). These TAP-tagged target proteins from yeast Saccharomyces cerevisiae allowed the protein to be isolated in two affinity purification steps. Subsequent structural analysis used the homobifunctional chemical crosslinker BS³ to join pairs of lysine residues on the surface of the purified protein via a flexible spacer arm. Mass spectrometry (MS) analysis of the crosslinked protein generated a set of mass values for crosslinked and non-crosslinked peptides, which was used to identify surface lysine residues in close proximity. The Automatic Spectrum Assignment Program was used to assign sequence information to the crosslinked peptides. This data provided inter-residue distance constraints that can be used to validate or refute theoretical protein structure models generated by structure prediction software such as SWISS-MODEL and RAPTOR. This approach was able to validate the structure models for four of the target proteins, TKL1, IPP1, HIS7 and ENO1. It also successfully selected the correct models for TKL1 and IPP1 from a protein model library and provided weak support for the HIS7, PGK1 and ENO1 models.

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.

Orientador: Fábio Cesar Gozzo
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: A Stanniocalcina-1 (STC1) é um hormônio glicoproteico que apresenta padrão de expressão diferencial destacado em diversas patologias, notadamente em neoplasias, mas seus aspectos funcionais e estruturais são pouco explorados até o momento. Nesse sentido, a STC1 foi escolhida como alvo para a utilização de uma abordagem integrativa das técnicas que utilizam os reagentes de ligação cruzada, espectrometria de massas e modelagem molecular para a modelagem estrutural. A partir dos experimentos de ligação cruzada, foram obtidas 37 restrições de distância envolvendo espécies ligadas com DSS, sendo 11 destas espécies com N-terminal e uma restrição envolvendo a espécie dimérica, além das cinco ligações de dissulfeto já publicadas. Essas restrições foram utilizadas para a geração de modelos estruturais nas plataformas online I-Tasser e Quark e, localmente, mais de 100.000 modelos pelo protocolo Ab Initio Relax do software Rosetta em quatro diferentes condições iniciais de modelagem. O Rosetta apresentou maior eficiência na geração de modelos quando ausente arquivo de predição de estrutura secundária. As restrições de distância foram ferramenta discriminatória fundamental para a seleção de estruturas candidatas para a STC1. O agrupamento utilizando o parâmetro global distante test (gdt) das 1500 modelos de menor score que respeitavam todas as restrições identificou 22 estruturas representativas estruturalmente distintas. Essas estruturas representativas podem agora ser utilizadas em testes envolvendo substituição molecular nos dados de difração de raios-X
Abstract: The Stanniocalcin-1 (STC1) is a glycoproteic hormone, which shows a differential expression pattern in a variety of pathologies, especially in neoplasia, but its functional and structural aspects have not been explored. Accordingly, the STC1 was chosen as a target to the use of an integrative approach including chemical cross-linking, mass spectrometry and molecular modeling. From cross-linking experiments,37 distance constrains were identified involving the DSS cross-linker, 11 of them in the N-terminus part of the protein and one involving the dimeric specie, in addition to five disulfide bonds already published. These constrains were used to generate structural models by I-Tasser and Quark online platforms and, locally, more than 100,000 models in the Ab Initio Relax protocol package present in the Rosetta software in four different modeling conditions. Rosetta was the most efficient in generating models when secondary structure prediction was absent. The distance constrains were found to be a key discriminatory tool for the selection of candidate structures for the STC1. For the 1,500 lowest score structures that satisfied the distance constrains, the clustering method employing the global distance test parameter (gdt) identified 22 structurally distinct representative structures. These representative structures can be used to in molecular replacement test to solve the X-Ray diffraction data
Mestrado
Quimica Organica
Mestre em Química

Le cancer de l'ovaire (OvCa) est le cancer le plus mortel parmi les cancers féminins. Il est souvent diagnostiqué tardivement ou mal diagnostiqué, ce qui le rend difficile à traiter. Les options de traitement incluent la chirurgie ou la chimiothérapie, toutefois la résistance à la chimiothérapie est un problème majeur. Il est donc urgent de trouver de nouvelles cibles et de développer de nouvelles stratégies pour surmonter cette résistance.Dans ce contexte le protéome fantôme est une source potentiellement riche de biomarqueurs. Le protéome fantôme, ou protéome alternatif, est composé de protéines traduites à partir de cadres de lecture ouverts alternatifs (AltORFs). Ces AltORFs proviennent de différents codons START issus de différente région de l'ARNm, tels qu'un décalage de cadre de lecture (+1, +2) dans la séquence codante de l'ADN (CDS), dans le 5'-UTR, 3'-UTR et éventuellement de la traduction d'ARN non codants (ncRNA).Les études sur les protéines alternatives (AltProts) sont souvent complexes et nécessite des études biomoléculaires coûteuses. Cependant, leurs fonctions peuvent être déduites en identifiant leurs partenaires d'interaction, la détection des interactions protéine-protéine (PPI) entre AltProts et protéines de référence (RefProts) peut aider à identifier leur fonction. La stratégie de pontage chimique (crosslink) combiné à la spectrométrie de masse (XL-MS) est un outil approprié à cet objectif. De plus, les outils bioinformatiques qui relient les informations fonctionnelles des RefProt et les analyses d'ontologie génique (GO) permettent la visualisation des voies de signalisation et le regroupement des RefProts en fonction de leur processus biologique, de leur fonction moléculaire ou de leur localisation cellulaire, et ainsi y placer certaine AltProt.Dans ce travail, nous avons développé une méthodologie combinant XL-MS et le fractionnement subcellulaire. L'étape de fractionnement subcellulaire nous a permis de réduire la complexité des échantillons analysés par chromatographie liquide et spectrométrie de masse (LC-HRMS/MS). Pour évaluer la validité des interactions, nous avons réalisé une modélisation moléculaire des structures 3D des AltProts, suivie d'une prédiction informatique de l'interaction et de mesure des distances de pontages identifiés expérimentalement. L'analyse a révélé des rôles d'AltProts dans les fonctions et les processus biologiques tel que la réparation de l'ADN ou encore la présentation d'antigène.La protéogénomique a été utilisée pour générer des bases de données protéiques personnalisées à partir des données de séquençage ARN afin d'étudier les protéomes de deux lignées cellulaires de cancer de l'ovaire (PEO-4 et SKOV-3) en comparaison avec une lignée cellulaire ovarienne normale (T1074). L'expression différentielle de plusieurs protéines a ainsi été identifiée entre les lignées cellulaires cancéreuses et normales, avec une association aux voies de signalisation connues pour le cancer. Des PPI ont également été identifiées dans les lignées cellulaires cancéreuses en utilisant la méthodologie XL-MS.Ce travail met en évidence le potentiel de l'approche protéogénomique pour découvrir de nouveaux aspects de la biologie du cancer de l'ovaire. Il nous permet d'identifier des protéines et des variants auparavant inconnus qui peuvent avoir une signification fonctionnelle. L'utilisation de bases de données protéiques personnalisées et de l'approche de réticulation a mis en lumière le "protéome fantôme", une vision du protéome restée inexplorée jusqu'à présent
Ovarian cancer (OvCa) has the highest mortality rate among female reproductive cancers worldwide. OvCa is often referred to as a stealth killer because it is commonly diagnosed late or misdiagnosed. Once diagnosed, OvCa treatment options include surgery or chemotherapy. However, chemotherapy resistance is a significant obstacle. Therefore, there is an urgent need to identify new targets and develop novel therapeutic strategies to overcome therapy resistance.In this context the ghost proteome is a potentially rich source of biomarkers. The ghost proteome, also known as the alternative proteome, consists of proteins translated from alternative open reading frames (AltORFs). These AltORFs originate from different start codons within mRNA molecules, such as the coding DNA sequence (CDS) in frameshifts (+1, +2), the 5'-UTR, 3'-UTR, and possible translation products from non-coding RNAs (ncRNA).Studies on alternative proteins (AltProts) are often limited due to their case-by-case occurrence and complexity. Obtaining functional protein information for AltProts requires complex and costly biomolecular studies. However, their functions can be inferred by profiling their interaction partners, known as "guilty by association" approaches. Indeed, assessing AltProts' protein-protein interactions (PPIs) with reference proteins (RefProts) can help identify their function and set them as research targets. Since there is a lack of antibodies against AltProts, crosslinking mass spectrometry (XL-MS) is an appropriate tool for this task. Additionally, bioinformatic tools that link protein functional information through networks and gene ontology (GO) analysis are also powerful. These tools enable the visualization of signaling pathways and the grouping of RefProts based on their biological process, molecular function, or cellular localization, thus enhancing our understanding of cellular mechanisms.In this work, we developed a methodology that combines XL-MS and subcellular fractionation. The key step of subcellular fractionation allowed us to reduce the complexity of the samples analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). To assess the validity of crosslinked interactions, we performed molecular modeling of the 3D structures of the AltProts, followed by docking studies and measurement of the corresponding crosslink distances. Network analysis indicated potential roles for AltProts in biological functions and processes. The advantages of this workflow include non-targeted AltProt identification and subcellular identification.Additionally, a proteogenomic analysis was performed to investigate the proteomes of two ovarian cancer cell lines (PEO-4 and SKOV-3 cells) in comparison to a normal ovarian epithelial cell line (T1074 cell). Using RNA-seq data, customized protein databases for each cell line were generated. Differential expression of several proteins, including AltProts, was identified between the cancer and normal cell lines. The expression of some RefProts and their transcripts were associated with cancer-related pathways. Moreover, the XL-MS methodology described above was used to identify PPIs in the cancerous cell lines.This work highlights the significant potential of proteogenomics in uncovering new aspects of ovarian cancer biology. It enables us to identify previously unknown proteins and variants that may have functional significance. The use of customized protein databases and the crosslinking approach have shed light on the "ghost proteome," an area that has remained unexplored until now

A novel protocol for the study of protein-nucleic acid interactions is presented and demonstrated to be feasible. The protocol combines photochemical crosslinking techniques and mass spectrometric methods into a new strategy for identifying protein domains or amino acid residues that are in close contact with nucleic acid in protein-nucleic acid complexes. Identifying nucleic acid binding domains in proteins provides a starting point for understanding structure-function relationships in protein-nucleic acid complexes. The protocol can be divided into three parts: 1) Cross linking of the protein-nucleic acid complex by irradiation with ultraviolet light and subsequently verifying the crosslinking by mass spectrometry; 2) Mass spectrometric peptide mapping of crosslinked protein-nucleic acid complexes to identify crosslinked peptide-nucleic acid hybrids; 3) Tandem mass spectrometric sequencing of peptide-nucleic acid hybrids to localize the crosslinked amino acid residue(s). The experimental data described in this dissertation documents our efforts to establish and implement this analytical protocol. Using several different protein-nucleic acid systems and different crosslinking techniques, we have demonstrated the feasibility of a mass spectrometric based approach to structurally characterize UV-crosslinked protein-nucleic acid complexes. Matrix-assisted laser desorption/ionization mass spectrometry was for the first time demonstrated to be highly effective for detection and molecular weight determination of intact, UV-crosslinked protein-nucleic acid complexes and for molecular weight determination of synthetic and UV-crosslinked peptide-nucleic acid hybrids. Electrospray ionization mass spectrometry and tandem mass spectrometry was demonstrated to be effective for analysis of synthetic peptide-nucleic acid hybrids and, in conjunction with HPLC, for peptide mapping of a protein. The first application of MALDI mass spectrometry to the characterization of crosslinked peptide-nucleic acid hybrids isolated from a photochemically crosslinked protein-nucleic acid complex demonstrate that the new protocol can be used to identify nucleic acid binding domains in proteins.
Graduation date: 1995

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in the presence of selected ligands: resting (no ligand), desensitized (saturating glycine) and open state (non-desensitizing ivermectin (IVM)-gated F207A/A288G GlyR). Photo-crosslinking methodology linked with mass spectrometric analysis was developed on systematically generated single Cys mutations in GlyR with both Cys null and IVM sensitive backgrounds to enable the study of state-dependent structures of GlyR in comparative crosslinking studies. Studies were conducted on A41C and H419C mutants. A41 is shown to be in proximity to the pre-M1 and the M2-M3 loop region crucial for gating. Prior to these studies, very little information on H419 was available as it is located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other related pentameric ligand-gated ion channels. These studies identified specific GlyR crosslinks unique to each conformational state and identified potential motions in the receptor upon gating and desensitization. The defined distance constraints will be used to update our model of human α1-GlyR and provide insight into channel function. Significantly, this methodological approach is amenable to study any allosteric protein and complement other high resolution structural studies in identifying protein dynamics.
Bayer School of Natural and Environmental Sciences;
Chemistry and Biochemistry
PhD;
Dissertation;

Mass spectrometry has critical roles in analytical chemistry, biomedical research and the diagnosis and treatment of human disease. Its ability to unambiguously identify and quantify a diverse range of biomolecules, from small molecule metabolites to large intact protein complexes in complex biological matrices, has solidified this technique’s place in the clinical chemists’ laboratory. However, mass spectrometry’s wider adoption by biochemists and clinicians is hindered by the inherent volume and complexity of the data it can generate from a biological context, and the subsequent level of specialist equipment and knowledge required to interpret results in a reliable and meaningful way. It is therefore apparent that a potential solution to this issue is to develop new methodologies, which adapt simple biochemical techniques into mass spectrometry sample preparation workflows that can be performed by biochemists using standard laboratory equipment. This thesis begins by reviewing some recent advances in clinical mass spectrometry and the current limitations of this technology. In doing so, we establish the importance of hyphenated techniques, which merge the concept of immunoassays with mass spectrometric detection to enable its adoption in high-throughput clinical and biomedical research applications. Many of these new approaches are only possible because of the development of efficient biocompatible chemical reactions, which facilitate the covalent modification of antibodies to improve their analysis via mass spectrometry. The concept of chemical labelling with mass tags, or synthetic linkers that fragment during ionisation and/or other stages of gas-phase analysis, has been widely explored as a strategy for detecting large heterogeneous biomolecules, such as antibodies. Chapter 2 explores the design, synthesis and application of an ultra-violet-cleavable linker for labelling intact antibodies to simplify their detection via matrix-assisted laser desorption/ionisation mass spectrometry. The novel aspect of this project is the utilisation of copper-catalysed azide-alkyne cycloaddition, or copper click chemistry, to enable straightforward modification of the detected photodissociation product, thereby offering the potential for multiplexed analysis of biomolecules. Despite the established utility of the copper click reaction, there are several limitations for its application in biological contexts. We therefore sought to identify alternative routes for modifying mass tags. Chapter 3 explores the utilisation of Diels-Alder cycloaddition chemistry for synthesising mass-tagged biomolecules, which conveniently facilitate efficient gas-phase fragmentation, even with the absence of a photocleavable moiety. The ability to easily detect proteins within biological specimens is incredibly valuable for diagnostics; however, the aetiology of many diseases involves modifications to proteins that occur after biosynthesis, or post-translational modifications. Chapter 4 explores the application of bottom-up proteomics methods to identify amino acid modifications to equine heart myoglobin in response to a model for oxidative stress, and characterise the products of its labelling with a novel fluorophore. Whereas the ability to analyse primary protein sequences and post-translational modifications is undoubtedly valuable, the function of many proteins is closely linked to their three-dimensional higher order structures, including transient interactions with other biomolecules. Reagents known as chemical crosslinkers, can be utilised to stabilise interactions between nearby amino acids prior to bottom-up proteomic analysis, thereby retaining tertiary and quaternary protein structural information. Chapter 5 describes some of the recent efforts from our research group to develop efficient strategies for synthesising and applying chemical cross-linkers with improved features for the purification and identification of cross-linked proteins. This thesis therefore explores various potential implementations of mass spectrometry for investigating the dynamic and complex roles of proteins in the context of human disease, from altered expression levels to post-translational modifications and protein-protein interactions.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2021

Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

The conversion of the native monomeric cellular prion protein (PrPC) into an aggregated pathological β-oligomeric (PrPβ) and an infectious form (PrPSc) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational change involved in this conversion are still unknown. My research hypothesis was that a specific structural rearrangement of normal PrPC monomers leads to the formation of new inter-subunit interaction interfaces in the prion aggregates, leading to aggregation. My approach was to use constraints obtained by structural proteomic methods to create a 3D model of urea-acid induced recombinant prion oligomers (PrPβ). My hypothesis was that this model would explain the mechanism of the conformational change involved in the conversion, the early formation of the β-structure nucleation site, and would describe the mode of assembly of the subunits within the oligomer. I applied a combination of limited proteolysis, surface modification, chemical crosslinking and hydrogen/deuterium exchange (HDX) with mass spectrometry for the differential characterization of the native and the urea-acid converted prion β-oligomer structures to get an insight into the mechanism of the conversion and aggregation. Using HDX, I detected a region of the protein in which backbone amides become more protected from exchange in PrPβ compared to PrPC. In order to obtain the inter-residue distance constraints to guide the assembly of the oligomer model, I then applied zero-length and short-range crosslinking to an equimolar mixture of 14N/15N-metabolically labeled β-oligomer thereby enabling the classification of the crosslinks as either intra-protein or inter-protein. Working with the Dokholyan group at the University of North Carolina at Chapel Hill, I was able to assemble a structure of the β-oligomer based on the combination of constraints obtained from all methods. By comparing the structures before and after the conversion, I was able to deduce the conformational change, that occurs during the conversion as the rearrangement and disassembly of the beta sheet 1– helix 1 – beta sheet 2 (β1-H1-β2) region from the helix 2 – helix 3 (H2-H3) core, forming new β-sheet nucleation site and resulting in the exposure of hydrophobic residues patches leading to formation of inter-protein contacts within aggregates.
Graduate
2018-06-14