Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers

Abstract

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (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 five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) 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, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands

“…major ampullate spidroin-encoding genes, MaSp1 and MaSp2 , have highest expression in major ampullate glands but are also expressed in tubuliform and anterior aggregate glands of L . hesperus ) confirming previous indications that not every spidroin paralog is restricted to a single gland type 33 – 36 . Both types of glue-producing aggregate glands are clearly distinguished from the fiber-forming glands by the paucity of spidroin production (Fig.…”

“…Other proteins known to form structural constituents of fibers include the Egg Case Proteins (ECP 1 & 2) 39 and Cysteine Rich Proteins (CRPs) 40 . We found ECPs were almost exclusively expressed in tubuliform glands, as expected, but CRPs, formerly only described from major ampullate silk 36 , 40 , were expressed in each of the individual gland types (Fig. 1b ) and are far more diverse than previously recognized.…”

“…Proteomic analyses of major ampullate fibers in L . hesperus found 15 of 35 identified proteins had no published homolog or no functional information 35 , 36 , further supporting this conclusion. It is also possible that some of the 209 new proteins are chaperones that assist with spidroin folding or aggregation.…”

Semi‐aquatic spider silks: transcripts, proteins, and silk fibres of the fishing spider, Dolomedes triton (Pisauridae)

“…Surprisingly, AcSp was also observed in tubuliform glands. Recent proteomic studies have detected AcSp proteins in the major ampullate glands of the cob-web weaver L. hesperus but not in tubuliform glands (Chaw et al, 2015;Larracas et al, 2016). AcSp gene expression has also been detected in the aggregate, flagelliform, and minor ampullate silk glands of the orb-web weaver N. clavipes, but not the tubuliform glands (Babb et al, 2017).…”

Web building and silk properties functionally covary among species of wolf spider

“…Spider MA silk comprises of a lipid-rich layer and a glycoprotein-rich skin covering a protein-based outer and inner core (Sponner et al, 2007;Papadopoulos et al, 2009;Heim et al, 2010;Blamires et al, 2017a). The core consists primarily of two types of proteins, called spidroins; major ampullate spidroin 1-type, or MaSp1-type proteins, and major ampullate spidroin 2type, or MaSp2-type proteins (Heim et al, 2010;Blamires et al, 2012aBlamires et al, ,b, 2016Larracas et al, 2016). The secondary structures of the spidroins have traditionally been thought to be critical for the silk's mechanical properties, with MaSp2-type being generally associated with greater extensibility and toughness and MaSp1type with greater ultimate strength (Hayashi et al, 1999).…”