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Characterization and identification of protein posttranslational modifications using protein enrichment and mass spectrometry

Wang, Liwen
http://rave.ohiolink.edu/etdc/view?acc_num=osu1234422949

Abstract Details

2009, Doctor of Philosophy, Ohio State University, Chemistry.
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.
Michael A. Freitas, PhD (Advisor)
Philip Grandinetti, PhD (Committee Member)
Thomas J. Magliery, PhD (Committee Member)
Pravin T.P. Kaumaya,, PhD (Committee Member)
296 p.
Analytical Chemistry
phosphorylation; mass spectrometry; bioinformatics; enrichments; chromatography

Recommended Citations

Citations

  • Wang, L. (2009). Characterization and identification of protein posttranslational modifications using protein enrichment and mass spectrometry [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1234422949

    APA Style (7th edition)

  • Wang, Liwen. Characterization and identification of protein posttranslational modifications using protein enrichment and mass spectrometry. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1234422949.

    MLA Style (8th edition)

  • Wang, Liwen. "Characterization and identification of protein posttranslational modifications using protein enrichment and mass spectrometry." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1234422949

    Chicago Manual of Style (17th edition)

osu1234422949
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© 2009, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.