John R. Engen
Mary Jo Ondrechen, Paul Vouros
Date of Award
Master of Science
Department or Academic Unit
College of Arts and Sciences. Department of Chemistry and Chemical Biology.
Pepsin, Proteolytic enzymes, Digestion
Pepsin is an aspartic acid protease that is commonly found in the stomach of many organisms. Porcine pepsin is the most studied and is fully active at pH 1.9 but inactive above pH ~7. Pepsin is known to have limited specificity and there are only general rules about its cleavage preferences. To further define rules regarding pepsin specificity, a database was constructed consisting of 40 proteins and 1344 peptide cleavages from the literature. Contemporary scientific literature was searched for all publications that involve pepsin digestion and mass spectrometry at pH 2.5-2.7. Peptide data for 40 proteins were extracted and combined to create a map of pepsin cleavage specificity. The frequency of cleavage for each protein was normalized based on how many times that specific combination of residues occurred in the protein sequence. In addition to the literature search, nine proteins along with E.coli whole cell lysate were digested at pH 1.0, 2.5 and 4.0. The proteins were analyzed with online pepsin digestion using an immobilized pepsin column and UPLC/ESI-MSE. The peptides and their fragments were identified with a combination of MSE, software analysis, and manual inspection. The analysis of the data indicated that pepsin maintains limited cleavage preferences. At pH 2.5, pepsin will cleave preferentially after most bulky, hydrophobic amino acids such as leucine and phenylalanine. Additionally, the residues that most often occur immediately following the cleaved peptide bond are tryptophan and tyrosine. It has also been shown that pepsin will rarely cleave at proline and histidine. Analysis performed at pH 1.0 and 4.0 yielded similar results.
Melissa H. Palashoff
Palashoff, Melissa H., "Determining the specificity of pepsin for proteolytic digestion" (2008). Chemistry Master's Theses. Paper 1. http://hdl.handle.net/2047/d10016636
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