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Protein Folding and Unfolding on the Millisecond Time Scale using Contained-Electrospray Ionization

Miller, Colbert
http://rave.ohiolink.edu/etdc/view?acc_num=osu1469026237

Abstract Details

2016, Master of Science, Ohio State University, Chemistry.
Top-down proteomics involves measuring the mass to charge ratio of an analyte protein without digestion. Unfolded proteins give better sequencing information and the workflow frequently involves a denaturing step before analysis. Manipulation of bulk solutions can be wasteful of valuable analyte solutions and can miss intermediates that describe the unfolding process. This thesis uses contained-electrospray ionization to perform online denaturing steps by mixing droplets in the electrospray plume with acidic vapors to decrease the pH of the droplet between the ESI emitter and the mass spectrometer inlet. The extent of protein denaturation can be controlled using different operational modes of the contained-electrospray ionization set-up. Operation mode Type I offers rapid protein denaturation where intermediated unfolding stages can be studied. For operation mode Type II, a cavity (5 mm) is created within the outer capillary which converts the electrospray droplets into thin liquid film and so increases the denaturation/reaction time for a more efficient unfolding. Operation modes Type I and Type II were tested using four proteins, myoglobin, ubiquitin, carbonic anhydrase, cytochrome C, with lysozyme as a control. Operation mode Type II was able to achieve higher average charge states using HCl vapor as a reagent than manipulating the bulk solution for myoglobin, ubiquitin, and cytochrome C. Lysozyme is unable to unfold without reducing disulfide bonds, and showed a minimal increase, indicating that changes are from proteins unfolding. In operation mode Type I myoglobin is highly charged with a heme group still attached, indicating that the reaction time is on the low millisecond to sub millisecond time scale. When the operation mode is switched to operation mode Type II, the heme completely falls off, indicating a longer reaction time. This thesis also introduces operation mode Type III, where the outer capillary of the contained-ES apparatus is replaced with a borosilicate theta capillary. This was used to test myoglobin, cytochrome c, and carbonic anhydrase II in 1% acetic acid with 80 mM ammonium acetate and 20 mM triethylammonium acetate to look at how the proteins refold when the pH is increased. All proteins showed a decrease in their average charge state. Charge states that were lower than what was detected in pure water were observed for both myoglobin and cytochrome C.
Abraham Badu-Tawiah (Advisor)
Vicki Wysocki (Committee Member)
59 p.
Chemistry
Protein Folding, Protein Unfolding, Millisecond Time Scale, Contained-Electrospray Ionization

Recommended Citations

Citations

  • Miller, C. (2016). Protein Folding and Unfolding on the Millisecond Time Scale using Contained-Electrospray Ionization [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469026237

    APA Style (7th edition)

  • Miller, Colbert. Protein Folding and Unfolding on the Millisecond Time Scale using Contained-Electrospray Ionization. 2016. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469026237.

    MLA Style (8th edition)

  • Miller, Colbert. "Protein Folding and Unfolding on the Millisecond Time Scale using Contained-Electrospray Ionization." Master's thesis, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469026237

    Chicago Manual of Style (17th edition)

osu1469026237
645
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This open access ETD is published by The Ohio State University and OhioLINK.