Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool

The presence of highly crosslinked protein network in hair and wool makes them very difficult substrates for protein extraction, a pre-requisite for further protein analysis and characterization. It is therefore imperative that these crosslinks formed by di-sulphide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea is commonly used as an efficient extractant. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex whereas phosphines, like Tris(2-carboxyethyl)phosphine (TCEP), remove material from the exocuticle. The relative extractability of the keratins and keratin associated proteins is also dependent on the concentration of chaotropes, reductants and pH, thus providing a means to preferentially extract these proteins. Ionic liquid such as 1-Butyl-3-methylimidazolium chloride is known to solubilize wool by disrupting non-covalent interactions, specifically intermolecular hydrogen bonds. 1-Butyl-3-methylimidazolium chloride proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry. Successful identification of proteins resolved by 1DE or 2DE and mass spectrometry (MS) is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher % sequence coverage, identification of unique peptides and higher score.