Membrane Digestion for Rapid Identification of Proteins Separated Using Capillary or Gel Electrophoresis

Doctoral Dissertation


This dissertation will describe rapid methods for identification of proteins separated under an electric field. The first project focuses on integrating a pepsin-containing membrane reactor into a capillary electrophoresis separation. Digestion of proteins occurs after their separation and just prior to mass spectrometry (MS). Thus, all the peptides from a given protein enter the mass spectrometer simultaneously to allow better characterization of proteins and provide more confident peptide identifications using their migration times. A custom-built sheath-flow interface contains the membrane to allow digestion just in front of the emitter tip. The second project focuses on identification of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The process uses electroblotting to transfer proteins from the gel to a capture membrane in a process related to a Western Blot. In lieu of an in-gel digest, proteolysis occurs in a trypsin-modified membrane as proteins transfer from the gel to the capture membrane. This work uses matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS to identify unknown proteins. Thus, unlike Western blotting, the technique does not require expensive antibodies or prior knowledge of the proteins of interest. In addition, a membrane modified with carbon nanotubes and polyelectrolytes replaced PVDF as the capture membrane. The conductivity of the membranes improves mass accuracy in MS, and the polyelectrolytes enhance capture efficiency. Overall, the new methods may help to rapidly identify proteins and their modifications.


Attribute NameValues
Author Kendall A. Ryan
Contributor Merlin L. Bruening, Research Director
Contributor Matt Champion, Committee Member
Contributor Jon Camden, Committee Member
Degree Level Doctoral Dissertation
Degree Discipline Chemistry and Biochemistry
Degree Name Doctor of Philosophy
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Defense Date
  • 2023-02-28

Submission Date 2023-03-08
Record Visibility Public
Content License
  • All rights reserved

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