ETD and ECD Mass Spectrometry Fragmentation for the Characterization of Protein Post Translational Modifications PDF Download

Author: Lisa Elviri
Publisher:
ISBN: 9789535101413
Category :
Languages : en
Pages :

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Author: John R. Griffiths
Publisher: John Wiley & Sons
ISBN: 1119250897
Category : Science
Languages : en
Pages : 377

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Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

Author: Huilin Li
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Author: Oscar Alzate
Publisher: CRC Press
ISBN: 1420076264
Category : Medical
Languages : en
Pages : 356

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In this, the post-genomic age, our knowledge of biological systems continues to expand and progress. As the research becomes more focused, so too does the data. Genomic research progresses to proteomics and brings us to a deeper understanding of the behavior and function of protein clusters. And now proteomics gives way to neuroproteomics as we beg

Author: Nicholas M. Riley
Publisher:
ISBN:
Category :
Languages : en
Pages : 409

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This dissertation presents research focusing on the development of new instrumentation and methodology to leverage ion-ion reactions for proteomic analyses. Electron transfer dissociation (ETD) technologies have proven a valuable alternative to collision-based fragmentation methods for sequencing peptides and proteins to advance global proteome characterization. Chapter 1 outlines the core concepts central to mass spectrometry (MS)-based proteomics, in addition to the basic principles of ETD and various strategies to improve its efficacy - including the technology that is the focus of this work, i.e., activated ion ETD (AI-ETD). Chapter 2 describes the first application of AI-ETD to intact proteins, which are more chemically complex and, thus, more difficult to sequence, than their peptide counterparts. Chapter 3 discusses a new strategy to improve signalto- noise in ETD spectra, which is especially beneficial for intact protein analysis and which has been incorporated into the newest generation of commercially available quadrupole-Orbitrap-linear ion trap hybrid MS systems. AI-ETD capabilities were also recently implemented on this stateof- the-art MS system (Chapter 4), and the ability to perform AI-ETD on this instrument enables comprehensive sequence coverage of moderately-sized intact proteins (Chapter 5), significantly improves proteoform characterization in large-scale analyses of complex mixtures of intact proteins (Chapter 6), and also enhances characterization of larger intact proteins (Chapter 7). Furthermore, AI-ETD improves characterization of post-translational modifications. Chapter 8 demonstrates the utility of AI-ETD for phosphosite localization in phosphopeptides and intact phosphoproteins, and Chapter 9 presents the largest glycoproteomic study to date by using AI-ETD to interrogate intact N-glycopeptides. Beyond positive-mode analyses of peptide and protein cations, ion-ion reactions also bring unique benefits to negative-mode analyses of precursor anions, where collision-based dissociation fails to consistently produce sequence-informative fragments. Chapter 10 describes implementation of negative ETD (NETD) and activated ion NETD (AI-NETD) and their application to whole-proteome sequencing in the negative mode, and Chapter 11 presents a modified search algorithm to improve interpretation of large-scale NETD and AI-NETD data. Conclusions and future directions of these projects are discussed in Chapter 12.

Author: Ying Ge
Publisher:
ISBN:
Category :
Languages : en
Pages : 354

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Author: Lisa Anne Vasicek
Publisher:
ISBN:
Category :
Languages : en
Pages : 338

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There continue to be great strides in the field of proteomics but as samples become more complex, the ability to increase sequence coverage and confidence in the identification becomes more important. Several methods of derivatization have been developed that can be used in combination with tandem mass spectrometry to identify and characterize proteins. Three types of activation, including infrared multiphoton dissociation, ultraviolet photodissociation, and electron transfer dissociation, are enhanced in this dissertation and compared to the conventional method of collisional induced dissociation (CID) to demonstrate the improved characterization of proteins. A free amine reactive phosphate group was synthesized and used to modify the N-terminus of digested peptides. This phosphate group absorbs at the IR wavelength of 10.6 [mu]m as well as the Vacuum-ultraviolet (VUV) due to an aromatic group allowing modified peptides to be dissociated by infrared multi-photon dissociation (IRMPD) or ultraviolet photodissociation (UVPD) whereas peptides without this chromophore are less responsive to IR or UV irradiation. The PD spectra for these modified peptides yield simplified MS/MS spectra due to the neutralization of all N-terminal product ions from the incorporation the negatively charged phosphate moiety. This is especially advantageous for UVPD due to the great number of product ions produced due to the higher energy deposition of the UV photons. The MS/MS spectra also produce higher sequence coverage in comparison to CID of the modified or unmodified peptides due to more informative fragmentation pathways generated upon PD from secondary dissociation and an increased ion trapping mass range. IRMPD is also implemented for the first time on an orbitrap mass spectrometer to achieve high resolution analysis of IR chromophore-derivatized samples as well as top-down analysis of unmodified proteins. High resolution/high mass accuracy analysis is extremely beneficial for characterization of complex samples due to the likelihood of false positives at lower resolutions/accuracies. For electron transfer dissociation, precursor ions in higher charge states undergo more exothermic electron transfer and thus minimize non-dissociative charge reduction. In this dissertation, cysteine side chains are alkylated with a fixed charge to deliberately increase the charge states of peptides and improve electron transfer dissociation. ETD can also be used to study protein structure by derivatizing the intact structure with a hydrazone reagent. A hydrazone bond will be preferentially cleaved during ETD facilitating the recognition of any modified residues through a distinguishing ETD fragmentation spectrum.

Author: Jeremy Lynn Balsbaugh
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Author: Guodong Chen
Publisher: Springer Science & Business Media
ISBN: 1441978623
Category : Science
Languages : en
Pages : 408

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This book highlights current approaches and future trends in the use of mass spectrometry to characterize protein therapies. As one of the most frequently utilized analytical techniques in pharmaceutical research and development, mass spectrometry has been widely used in the characterization of protein therapeutics due to its analytical sensitivity, selectivity, and specificity. This book begins with an overview of mass spectrometry techniques as related to the analysis of protein therapeutics, structural identification strategies, quantitative approaches, followed by studies involving characterization of process related protein drug impurities/degradants, metabolites, higher order structures of protein therapeutics. Both general practitioners in pharmaceutical research and specialists in analytical sciences will benefit from this book that details step-by-step approaches and new strategies to solve challenging problems related to protein therapeutics research and development.

Author: Liwen Wang
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Abstract: This dissertation describes a proteomic workflow for the analysis of protein post-translational modifications (PTMs). The workflow combines the techniques for protein enrichment, multi-dimensional separations, mass spectrometry (MS) and automatic data analysis. The workflow was developed to improve the application of proteomic analysis in the realms of biomarker discovery and experimental therapeutic research. Chapter 2 presents an immunoaffinity chromatography method that was developed to enrich acetylated histones. A self-packed immunoaffinity capillary column was developed using commercial antibodies that could be recycled and used for on-line and off-line enrichment. The acetylated fractions were collected and identified by Matrix Assisted Laser Desorption (MALDI) MS and electrospray ionization (ESI) liquid chromatography tandem mass spectrometry (LC-MS/MS). In chapter 3 an optimized phosphoproteomic analysis workflow based on phosphopeptide enrichment, data-dependant neutral loss mass spectrometry and a novel hierarchical database searching is described. The combination of these approaches improved the confidence of phosphopeptide identifications. Chapter 4 describes the use of phosphoprotein enrichment and a tandem phosphoprotein and phosphopeptide enrichment to improve the identification of phosphoproteins and localization of the phosphorylation sites. Purification of global phosphoproteins from primary CLL B-cells was conducted by use of PhosTag Zn2 enrichment strategy at neutral pH. SDS-PAGE gel was used to separate the purified phosphoprotein fraction and Pro-Q diamond staining was employed to visualize those phosphoprotein bands. Shot-gun proteomic analysis was then performed to identify all the enriched phosphoproteins in the gel. Phosphopeptide enrichment was used in tandem to map phosphorylation sites of the enriched phosphoproteins. Chapter 5 describes the identification of tyrosine phosphoproteins associated with immunotherapy of malignant cells with the small modular immunopharmaceutical targeted against CD37 (CD37-SMIPTM). This drug induces apoptosis and antibody-dependent cellular cytotoxicity (ADCC) in primary Chronic Lymphocyte Leukemia (CLL) cells. Tyrosine phosphorylation of proteins was investigated as an early activation event for the cytotoxicity. Immunoprecipitation was used to purify the phosphotyrosine proteins from treated cell lysate and untreated cell lysate. Detection of modulation of tyrosine phosphorylation and identification of those tyrosine phosphoproteins after treatment by proteomic approaches revealed proteins associated with the signaling pathway activated by immunotherapy. Chapter 6 describes a direct application of the proteomic platform developed in Chapter 3 combined with LC-MS protein profiling. The modulation of histone phosphorylation isoforms induced by various chemotherapy drugs was detected by LC-MS screening. We detected the dephosphorylation of histones H1 and hyperphosphorylation of H2A.X associated with the different drug treatments.