Spider silk is characterized to be incredibly strong and versatile, making it a valuable resource for manufacturing synthetic fibers for industrial applications. The objective of this study is to analyze the scaffolding fibers of Lactrodectus hesperus, a cobweaver, which creates asymmetrical three-dimensional web. To date, scaffolding silk has been shown to contain two structural silk proteins, Major Ampullate Spidroin 1 (MaSp1) and Major Ampullate Spidroin 2 (MaSp2). We hypothesize that scaffolding silk contains other novel proteins that have yet to be identified. In order to explore the potential of novel proteins assembled into scaffolding fibers, we collected threads from female black widow spiders, dissolved the fibers in a protein denaturant, alkylated the side chain cysteine residues, and then digested the protein mixture with trypsin. Tryptic peptides were separated using nano high performance liquid chromatography (nanoHPLC) and then subject to MS and MS/MS analysis using an Orbitrap Fusion mass spectrometer. Analysis of the scaffolding threads revealed the presence of several new proteins in scaffolding fibers
