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Date of Award

2017

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Craig Vierra

First Committee Member

Geoffrey Lin-Cereghino

Second Committee Member

Lisa Wrischnik

Abstract

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, MaSp1 and MaSp2. Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using 5 different solvents, followed by nanoLC-MS/MS analysis with an Orbitrap Fusion™ Tribrid™ mass spectrometer. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included CID, HCD, and ETD to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin AcSp1 in dragline silk, a spidroin widely recognized as the major constituent of wrapping silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least 7 common proteins: major ampullate spidroin 1 and 2 (MaSp1 and MaSp2), 3 members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), and two uncharacterized proteins, CRISP3 and fasciclin. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers

Pages

66

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