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Pavel A. Pevzner - One of the best experts on this subject based on the ideXlab platform.
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automated de novo Protein Sequencing of monoclonal antibodies
Nature Biotechnology, 2008Co-Authors: Nuno Bandeira, Pavel A. Pevzner, Victoria Pham, David Arnott, Jennie R LillAbstract:De novo Protein Sequencing of monoclonal antibodies is required when the cDNA or the original cell line is not available, or when characterization of posttranslational modifications is needed to verify antibody integrity and effectiveness. We demonstrate that Comparative Shotgun Protein Sequencing (CSPS) based on tandem mass spectrometry can reduce the time required to sequence an antibody to 72 hours, a dramatic reduction as compared to the classical technique of Edman degradation. We therefore argue that CSPS has the potential to be a disruptive technology for all Protein Sequencing applications.
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Shotgun Protein Sequencing Assembly of Peptide Tandem Mass Spectra from Mixtures of Modified Proteins
Molecular & cellular proteomics : MCP, 2007Co-Authors: Nuno Bandeira, Karl R. Clauser, Pavel A. PevznerAbstract:Despite significant advances in the identification of known Proteins, the analysis of unknown Proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for Sequencing of unknown Proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown Proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA Sequencing strategies have not been extended to automated Protein Sequencing. We demonstrate, for the first time, the feasibility of automated shotgun Protein Sequencing of Protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various Proteins in western diamondback rattlesnake venom. We further argue that shotgun Protein Sequencing has the potential to overcome the limitations of current Protein Sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown Proteins.
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WABI - Shotgun Protein Sequencing
Lecture Notes in Computer Science, 2007Co-Authors: Pavel A. PevznerAbstract:Despite significant advances in the identification of known Proteins, the analysis of unknown Proteins by tandem mass spectrometry (MS/MS) still remains a challenging open problem. Although Klaus Biemann recognized the potential of mass spectrometry for Sequencing of unknown Proteins in the 1980s, low-throughput Edman degradation followed by cloning still remains the main method to sequence unknown Proteins. The automated spectral interpretation has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed, the powerful Shotgun DNA Sequencing strategies have not been extended to Protein Sequencing yet.We demonstrate, for the first time, the feasibility of Shotgun Protein Sequencing of Protein mixtures and validate this approach by generating highly accurate de novo reconstructions of various Proteins in western diamondback rattlesnake venom. We further argue that Shotgun Protein Sequencing has the potential to overcome the limitations of current Protein Sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown Proteins. We further describe applications of this technique to analyzing Proteins that are not directly inscribed in DNA sequences (like antibodies and fusion Proteins in cancer). This is a joint work with Nuno Bandeira (UCSD) and Karl Clauser (Broad).
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Shotgun Protein Sequencing by tandem mass spectra assembly.
Analytical chemistry, 2004Co-Authors: Nuno Bandeira, Haixu Tang, Vineet Bafna, Pavel A. PevznerAbstract:The analysis of mass spectrometry data is still largely based on identification of single MS/MS spectra and does not attempt to make use of the extra information available in multiple MS/MS spectra from partially or completely overlapping peptides. Analysis of MS/MS spectra from multiple overlapping peptides opens up the possibility of assembling MS/MS spectra into entire Proteins, similarly to the assembly of overlapping DNA reads into entire genomes. In this paper, we present for the first time a way to detect, score, and interpret overlaps between uninterpreted MS/MS spectra in an attempt to sequence entire Proteins rather than individual peptides. We show that this approach not only extends the length of reconstructed amino acid sequences but also dramatically improves the quality of de novo peptide Sequencing, even for low mass accuracy MS/MS data.
Nuno Bandeira - One of the best experts on this subject based on the ideXlab platform.
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Shotgun Protein Sequencing with Meta-contig Assembly
Molecular & cellular proteomics : MCP, 2012Co-Authors: Adrian Guthals, Karl R. Clauser, Nuno BandeiraAbstract:Full-length de novo Sequencing from tandem mass (MS/MS) spectra of unknown Proteins such as antibodies or Proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low Sequencing accuracy. Our shotgun Protein Sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous Proteins to group smaller contig sequences into full-length Protein sequences, thus limiting its effectiveness on sequences from poorly annotated Proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous Protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo Sequencing limitations relate to MS/MS acquisition settings.
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automated de novo Protein Sequencing of monoclonal antibodies
Nature Biotechnology, 2008Co-Authors: Nuno Bandeira, Pavel A. Pevzner, Victoria Pham, David Arnott, Jennie R LillAbstract:De novo Protein Sequencing of monoclonal antibodies is required when the cDNA or the original cell line is not available, or when characterization of posttranslational modifications is needed to verify antibody integrity and effectiveness. We demonstrate that Comparative Shotgun Protein Sequencing (CSPS) based on tandem mass spectrometry can reduce the time required to sequence an antibody to 72 hours, a dramatic reduction as compared to the classical technique of Edman degradation. We therefore argue that CSPS has the potential to be a disruptive technology for all Protein Sequencing applications.
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Shotgun Protein Sequencing Assembly of Peptide Tandem Mass Spectra from Mixtures of Modified Proteins
Molecular & cellular proteomics : MCP, 2007Co-Authors: Nuno Bandeira, Karl R. Clauser, Pavel A. PevznerAbstract:Despite significant advances in the identification of known Proteins, the analysis of unknown Proteins by MS/MS still remains a challenging open problem. Although Klaus Biemann recognized the potential of MS/MS for Sequencing of unknown Proteins in the 1980s, low throughput Edman degradation followed by cloning still remains the main method to sequence unknown Proteins. The automated interpretation of MS/MS spectra has been limited by a focus on individual spectra and has not capitalized on the information contained in spectra of overlapping peptides. Indeed the powerful shotgun DNA Sequencing strategies have not been extended to automated Protein Sequencing. We demonstrate, for the first time, the feasibility of automated shotgun Protein Sequencing of Protein mixtures by utilizing MS/MS spectra of overlapping and possibly modified peptides generated via multiple proteases of different specificities. We validate this approach by generating highly accurate de novo reconstructions of multiple regions of various Proteins in western diamondback rattlesnake venom. We further argue that shotgun Protein Sequencing has the potential to overcome the limitations of current Protein Sequencing approaches and thus catalyze the otherwise impractical applications of proteomics methodologies in studies of unknown Proteins.
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Shotgun Protein Sequencing by tandem mass spectra assembly.
Analytical chemistry, 2004Co-Authors: Nuno Bandeira, Haixu Tang, Vineet Bafna, Pavel A. PevznerAbstract:The analysis of mass spectrometry data is still largely based on identification of single MS/MS spectra and does not attempt to make use of the extra information available in multiple MS/MS spectra from partially or completely overlapping peptides. Analysis of MS/MS spectra from multiple overlapping peptides opens up the possibility of assembling MS/MS spectra into entire Proteins, similarly to the assembly of overlapping DNA reads into entire genomes. In this paper, we present for the first time a way to detect, score, and interpret overlaps between uninterpreted MS/MS spectra in an attempt to sequence entire Proteins rather than individual peptides. We show that this approach not only extends the length of reconstructed amino acid sequences but also dramatically improves the quality of de novo peptide Sequencing, even for low mass accuracy MS/MS data.
Michael Kinter - One of the best experts on this subject based on the ideXlab platform.
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Protein Sequencing with tandem mass spectrometry.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Assem G. Ziady, Michael KinterAbstract:The recent introduction of electrospray ionization techniques that are suitable for peptides and whole Proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for Proteins. The advantages gained by these approaches over traditional Edman Degradation Sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify Proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby Protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the Protein. This powerful technique has been instrumental for the study of Proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of Protein expression in cystic fibrosis as an example.
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Protein Sequencing and identification using tandem mass spectrometry
2000Co-Authors: Michael Kinter, Nicholas E ShermanAbstract:An Introduction to Protein Sequencing Using Tandem Mass Spectrometry The Primary Structure of Proteins and a Historical Overview of Protein Sequencing Fundamental Mass Spectrometry Collisionally Induced Dissociation of Protonated Peptide Ions and the Interpretation of Product Ion Spectra Basic Polyacrylamide Gel Electrophoresis The Preparation of Protein Digests for Mass Spectrometric Sequencing Experiments Mass Spectrometric Analysis of Tryptic Digests Protein Identification by Database Searching Sequence Analysis of Novel Proteins The Characterization of Post-Translationally Modified Proteins Using Tandem Mass Spectrometry Index.
Kira Vyatkina - One of the best experts on this subject based on the ideXlab platform.
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De Novo Protein Sequencing by Combining Top-Down and Bottom-Up Tandem Mass Spectra
Journal of proteome research, 2014Co-Authors: Xiaowen Liu, Lennard J. M. Dekker, Martijn M. Vanduijn, Theo M. Luider, Nikola Tolić, Qiang Kou, Mikhail Dvorkin, Sonya Alexandrova, Kira VyatkinaAbstract:There are two approaches for de novo Protein Sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel Protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the Protein. Because each tandem mass spectrum covers only a short peptide of the target Protein, the key to high coverage Protein Sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole Proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a Protein. We propose an algorithm, TBNovo, for de novo pro...
John P. Marino - One of the best experts on this subject based on the ideXlab platform.
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Leveraging nature’s biomolecular designs in next-generation Protein Sequencing reagent development
Applied Microbiology and Biotechnology, 2020Co-Authors: Jennifer Tullman, John P. Marino, Zvi KelmanAbstract:Next-generation approaches for Protein Sequencing are now emerging that could have the potential to revolutionize the field in proteomics. One such Sequencing method involves fluorescence-based imaging of immobilized peptides in which the N-terminal amino acid of a polypeptide is readout sequentially by a series of fluorescently labeled biomolecules. When selectively bound to a specific N-terminal amino acid, the NAAB ( N -terminal amino acid binder) affinity reagent identifies the amino acid through its associated fluorescence tag. A key technical challenge in implementing this fluoro-Sequencing approach is the need to develop NAAB affinity reagents with the high affinity and selectivity for specific N-terminal amino acids required for this biotechnology application. One approach to develop such a NAAB affinity reagent is to leverage naturally occurring biomolecules that bind amino acids and/or peptides. Here, we describe several candidate biomolecules that could be considered for this purpose and discuss the potential for developability of each. Key points • Next-generation Sequencing methods are emerging that could revolutionize proteomics. • Sequential readout of N-terminal amino acids by fluorescent-tagged affinity reagents. • Native peptide/amino acid binders can be engineered into affinity reagents. • Protein size and structure contribute to feasibility of reagent developability.
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Leveraging nature's biomolecular designs in next-generation Protein Sequencing reagent development.
Applied microbiology and biotechnology, 2020Co-Authors: Jennifer Tullman, John P. Marino, Zvi KelmanAbstract:Next-generation approaches for Protein Sequencing are now emerging that could have the potential to revolutionize the field in proteomics. One such Sequencing method involves fluorescence-based imaging of immobilized peptides in which the N-terminal amino acid of a polypeptide is readout sequentially by a series of fluorescently labeled biomolecules. When selectively bound to a specific N-terminal amino acid, the NAAB (N-terminal amino acid binder) affinity reagent identifies the amino acid through its associated fluorescence tag. A key technical challenge in implementing this fluoro-Sequencing approach is the need to develop NAAB affinity reagents with the high affinity and selectivity for specific N-terminal amino acids required for this biotechnology application. One approach to develop such a NAAB affinity reagent is to leverage naturally occurring biomolecules that bind amino acids and/or peptides. Here, we describe several candidate biomolecules that could be considered for this purpose and discuss the potential for developability of each.
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Strategies for Development of a Next-Generation Protein Sequencing Platform
Trends in biochemical sciences, 2019Co-Authors: Nicholas Callahan, Jennifer Tullman, Zvi Kelman, John P. MarinoAbstract:Proteomic analysis can be a critical bottleneck in cellular characterization. The current paradigm relies primarily on mass spectrometry of peptides and affinity reagents (i.e., antibodies), both of which require a priori knowledge of the sample. An unbiased Protein Sequencing method, with a dynamic range that covers the full range of Protein concentrations in proteomes, would revolutionize the field of proteomics, allowing a more facile characterization of novel gene products and subcellular complexes. To this end, several new platforms based on single-molecule Protein-Sequencing approaches have been proposed. This review summarizes four of these approaches, highlighting advantages, limitations, and challenges for each method towards advancing as a core technology for next-generation Protein Sequencing.
