Date of Award

2014

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Scudder, Paul

Keywords

Proteomics, Proteases, Mass Spectrometry

Area of Concentration

Biochemistry

Abstract

Proteomics, the study of the protein functional complement of the genome, deals with similar issues of amino-acid sequence coverage and depth. Trypsin has been the most common protease used to cleave peptides into manageable units due the many optimum traits of tryptic digestions for mass spectrometry (MS) analyses for the entire history of “shotgun” proteomics. Instrumental and computational methods for proteomic analyses have been tailored for tryptic peptides as a result. This project explored the feasibility of an alternative approach that utilizes a sequential digestion by lesser-used proteases, a C-terminal arginine peptidase (Arg-C or Clostripain) and a specific N-terminal lysine metallopeptidase (Lys-N). Lys-N was isolated from Grifola frondosa (maitake), and Arg-C was obtained from a commercial source. Doubly-digested peptides from a mouse brain protein extract were separated by offline strong cation exchange (SCX) chromatography followed by on-line ultra-performance liquid chromatography (UPLC) and analysis by high resolution mass spectrometry. This preparation produced a population of unique peptides that were tightly retained on the SCX column and ionized well by electrospray ionization (ESI). Peptides were identifiable via extracted ion tracking as well as the SEQUEST search algorithm. Thus, the method described herein represents a potential alternate route to acquire complementary data to conventional proteomic approaches.

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