Characterization of Photochemically Cross-linked Protein-nucleic Acid Complexes by Mass Spectrometry PDF Download
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Author: Ole Nørregaard Jensen
Publisher:
ISBN:
Category : Nucleic acids
Languages : en
Pages : 318
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Book Description
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.
Author: Ole Nørregaard Jensen
Publisher:
ISBN:
Category : Nucleic acids
Languages : en
Pages : 318
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Book Description
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.
Author: Luisa Welp
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Protein-nucleic acid interactions are a key part of essential cellular processes and their disturbance often results in the development of diseases. Crosslinking mass spectrometry (XL-MS)-based approaches have become increasingly popular for the proteome-wide discovery of nucleic acid-binding proteins. The basic principle of the methodology relies on covalent attachment of proteins and binding nucleic acids by crosslinking, which makes the heteroconjugates amenable to MS analysis. Building up on comprehensive nucleic acid-binding protein inventories, there is an increasing demand for higher...
Author: Catalin E. Doneanu
Publisher:
ISBN:
Category : DNA-protein interactions
Languages : en
Pages : 302
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Book Description
Author: Shan S. Wong
Publisher: CRC Press
ISBN: 084937491X
Category : Medical
Languages : en
Pages : 624
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Book Description
Since the publication of the first edition of Chemistry of Protein Conjugation and Cross-Linking in 1991, new cross-linking reagents, notably multifunctional cross-linkers, have been developed and synthesized. The completion of the human genome project has opened a new area for studying nucleic acid and protein interactions using nucleic acid cross-linking reagents, and advances have also been made in the area of biosensors and microarray biochips for the detection and analysis of genes, proteins, and carbohydrates. In addition, developments in physical techniques with unprecedented sensitivity and resolution have facilitated the analysis of cross-linked products. Updated to reflect the advances of the 21st century, this book offers: An overview of the chemical principles underlying the processes of cross-linking and conjugation A thorough list of cross-linking reagents published in the literature since the first edition, covering monofunctional, homobifunctional, heterobifunctional, multifunctional, and zero-length cross-linkers Reviews of the use of these reagents in studying protein tertiary structures, geometric arrangements of subunits within complex proteins and nucleic acids, near-neighbor analysis, protein-to-protein or ligand–receptor interactions, and conformational changes of biomolecules Discusses the application of immunoconjugation for immunoassays, immunotoxins for targeted therapy, microarray technology for analysis of various biomolecules, and solid state chemistry for immobilizations
Author: Joseph H Banoub
Publisher: CRC Press
ISBN: 9780367384685
Category :
Languages : en
Pages : 504
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Book Description
Assembling the work of an international panel of researchers, Mass Spectrometry of Nucleosides and Nucleic Acids summarizes and reviews the latest developments in the field and provides a window on the next generation of analysis. Beginning with an overview of recent developments, the book highlights the most popular ionization methods and illustrates the diversity of strategies employed in the characterization and sequencing of DNA and RNA oligomers, nucleosides, nucleotides, and adducts. It describes studies performed on deoxyinosine and its analogues and provides an introduction to tandem mass spectrometry (MS/MS). Next, the contributors examine mass spectrometric application in the study of cyclic nucleotides in biochemical signal transduction. They analyze urinary modified nucleosides and explore DNA adducts. They discuss isotope labeling of DNA-mass spectrometry (ILD-MS) and examine various uses of electrospray ionization mass spectrometry (ESI-MS). The book reviews recent progress in the direct MS characterization of noncovalent nucleic acid-protein complexes, explores the interaction and ionization of guanidine-derived compounds with highly acidic biomolecules, and examines quantitative identification of nucleic acids via signature digestion products detected using mass spectrometry. The book describes a direct-infusion ESI-MS approach that can serve as a screening technique for the presence of modified nucleosides from small RNAs. Lastly, it discusses the LC-MS/MS method for the in vitro replication studies on damage-containing DNA substrates, and concludes with an examination of the influence of metal ions on the structure and reactivity of nucleic acids. The exciting developments in mass spectrometry technology have fueled incredible advances in our understanding of nucleic acids and their complexes. The contributions presented in this volume capture the range of these advances, helping to inspire new findings a
Author: Katrin Marcus
Publisher: Humana Press
ISBN: 9781617798849
Category : Science
Languages : en
Pages : 539
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Book Description
Protein modifications and changes made to them, as well as the quantities of expressed proteins, can define the various functional stages of the cell. Accordingly, perturbations can lead to various diseases and disorders. As a result, it has become paramount to be able to detect and monitor post-translational modifications and to measure the abundance of proteins within the cell with extreme sensitivity. While protein identification is an almost routine requirement nowadays, reliable techniques for quantifying unmodified proteins (including those that escape detection under standard conditions, such as protein isoforms and membrane proteins) is not routine. Quantitative Methods in Proteomics gives a detailed survey of topics and methods on the principles underlying modern protein analysis, from statistical issues when planning proteomics experiments, to gel-based and mass spectrometry-based applications. The quantification of post-translational modifications is also addressed, followed by the “hot” topics of software and data analysis, as well as various overview chapters which provide a comprehensive overview of existing methods in quantitative proteomics. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Quantitative Methods in Proteomics serves as a comprehensive and competent overview of the important and still growing field of quantitative proteomics.
Author: Eugen Netz
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Proteins are the most active molecules in living bodies. They catalyze chemical reactions, provide structural support for cells and allow organisms to move. Their function is intrinsically linked to their folded structure. Resolving the structures of proteins and protein complexes is crucial for our understanding of basic biological processes and diseases. Cross-Linking Mass Spectrometry (XL-MS) is a method to gain structural insights into protein complexes. The field of XL-MS data analysis software is not yet as established as many other methods in proteomics. XL-MS analysis software has significant room for improvement in terms of sensitivity, efficiency and standardization of file formats and workflows to facilitate interoperability and reproducibility. In this thesis we present a new XL-MS search engine, OpenPepXL. We develop an algorithm that scores all candidate cross-linked peptide pairs and is efficient enough to be used on a standard desktop PC for most applications. OpenPepXL supports the standardized XL-MS identification file format defined as a part of the MzIdentML 1.2 specifications that were developed in collaboration with the Proteomics Standards Initiative. We benchmark OpenPepXL against other state-of-the-art XL-MS identification tools on multiple datasets that allow cross-link validation through structures or other means. We show that our exhaustive approach, although not the quickest one, is superior in sensitivity to other tools. We suggest this is due to some tools improving their processing time by discarding too many candidates in early steps of the data analysis. We apply XL-MS analysis with OpenPepXL to multiple protein complexes related to meiosis and the type III secretion system. The first project involved several proteins with unknown structures, some of which are expected to be at least partially intrinsically disordered and therefore difficult to investigate using most traditional structural research methods. Unfortunately, we could not find cross-links between the interaction sites we were interested in the most, but we were able to identify many others in these complexes and gained some structural insights. In the second project we used the photo-cross-linking amino acid pBpa to test very specific hypotheses about interactions within the type III secretion system. We were not able to gain any new structural information yet. However, we could confirm that this is a viable approach. It is possible to identify cross-links between a pBpa residue incorporated into a protein sequence and a residue it cross-links to on a residue level resolution.
Author: Alex Huszagh
Publisher:
ISBN: 9780355414288
Category :
Languages : en
Pages : 139
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Book Description
Cross-linking mass spectrometry maps the structural topology of protein complexes by using the distance between linked residues as spatial constraints, complementing other structural biology techniques. However, the identification of cross-linked peptides scales poorly with the number of proteins analyzed. Our lab has previously developed MS-cleavable cross-linkers to enable the separation of cross-linked peptides prior to sequencing, enabling peptide identifica- tion using standard peptide search databases. We describe the design and implementation of platform and application named XLTools for the automated identification of MS-cleavable cross-linked peptides. XLTools supports open and proprietary data formats and common peptide search databases, facilitating its integration into existing workflows. Furthermore, we developed peak-picking and validation algorithms to enable the accurate quantitation of cross-linked peptides in complex samples. We demonstrate the application of XLTools to the quantitative analysis of the 26S proteasome cross-linked in vivo and in vitro.
Author: Dallas Allan Connor
Publisher:
ISBN:
Category : DNA-protein interactions
Languages : en
Pages : 252
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Book Description
Author: Franz Hillenkamp
Publisher: John Wiley & Sons
ISBN: 3527673741
Category : Science
Languages : en
Pages : 447
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Book Description
This authoritative book on MALDI MS, now finally available in its second edition and edited by one of its inventors, gives an in-depth description of the many different applications, along with a detailed discussion of the technology itself. Thoroughly updated and expanded, with contributions from key players in the field, this unique book provides a comprehensive overview of MALDI MS along with its possibilities and limitations. The initial chapters deal with the technology and the instrumental setup, followed by chapters on the use of MALDI MS in protein research (including proteomics), genomics, glycomics and lipidomics. The option of MALDI-MS for the analysis of polymers and small molecules are also covered in separate chapters, while new to this edition is a section devoted to the interplay of MALDI MS and bioinformatics. A much-needed practical and educational asset for individuals, academic institutions and companies in the field of bioanalytics.
