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Analysis of Protein Three-Dimensional Structures and Capture of Organic Reaction Intermediates by Mass Spectrometry

Zheng, Qiuling
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1457995358

Abstract Details

2016, Doctor of Philosophy (PhD), Ohio University, Chemistry and Biochemistry (Arts and Sciences).
Chemical cross-linking (CL) combined with mass spectrometry (MS) has become a powerful method to provide low-resolution protein structural information. However, the identification of cross-linked peptides and the complexity of their fragmentation patterns hamper the development of this method. In this dissertation, online coupling of electrochemistry (EC) with liquid sample desorption electrospray ionization (DESI)-MS is developed for rapid identification of cross-linked peptides by introducing a disulfide bond-containing cross-linker, dithiobis[succinimidyl propionate] (DSP). Due to the electrolytic response of disulfide bonds, cross-links are subject to pronounced intensity decreases upon electrolytic reduction, suggesting a new way to identify cross-links. In addition, mass shifts before and after electrolysis suggests the linkage pattern of the cross-links. On this basis of novel method, isotope labeled chemical CL strategy is also developed for differentiating cross-link products from native disulfide bonds. More importantly, light and heavy isotope labeled cross-linkers (i.e., DSP-d0 and DSP-d8) can be used to react with proteins in two different conformational states separately and the modified proteins are merged in equal molar ratio before enzymatic digestion and MS analysis. Such an analysis can be useful to probe protein conformational changes. Our experimental results show that EC-assisted chemical CL MS method is rapid and no need for chromatographic separation, which would be of high value for structural proteomics research. In another project, MS is used to capture transient organometallic reaction intermediates. Palladium-mediated cross-coupling reactions to form C-C, C-N, C-O, and C-S bonds are one of the most powerful organometallic transformations employed in organic synthesis. Evidence has been provided that the formation of presumed active catalyst Pd0, which has been regarded as the true reaction intermediate in various Pd-catalyzed coupling reactions, ensures the success of coupling reactions. However, due to its high reactivity, the short-lived Pd0 intermediates have not been experimentally identified. Herein, monophosphine-ligated Pd0 intermediates generated from various precatalysts are directly captured and detailed structural information is provided by DESI-MS. Thus, the successful capture and characterization of Pd0 intermediates suggest the power of DESI in detecting short-lived and difficult-to-study reactive intermediates and benefits the reaction mechanism studies.
Hao Chen (Advisor)
Analytical Chemistry
Mass spectrometry; electrochemistry; chemical cross-linking; reaction intermediates

Recommended Citations

Citations

  • Zheng, Q. (2016). Analysis of Protein Three-Dimensional Structures and Capture of Organic Reaction Intermediates by Mass Spectrometry [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1457995358

    APA Style (7th edition)

  • Zheng, Qiuling. Analysis of Protein Three-Dimensional Structures and Capture of Organic Reaction Intermediates by Mass Spectrometry. 2016. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1457995358.

    MLA Style (8th edition)

  • Zheng, Qiuling. "Analysis of Protein Three-Dimensional Structures and Capture of Organic Reaction Intermediates by Mass Spectrometry." Doctoral dissertation, Ohio University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1457995358

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Ohio University and OhioLINK.