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    Biomaterials

    Mass Spectrometry Identification of HIC-Purified Human Hair Keratins

    (C-114) Mass Spectrometry Identification of HIC-Purified Human Hair Keratins

    Friday, October 13, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row C - Poster # 114
    Introduction:: Introduction:  Keratins are intermediate filament proteins abundantly present in human hair, usually in highly-organized multimers.  These structures are often coiled and interconnected together with disulfide bonds facilitated by the numerous cysteine residues.  Accordingly, these network-forming keratins can be employed and engineered as scaffolds for cell and tissue growth and drug-delivery.  As these biomaterials are increasingly studied, the separation and identification of specific keratin types is often overlooked but very important in downstream applications since human hair extract contains a heterogenous mix of about a dozen different hair keratins with smaller molecule keratin associated proteins (KAPs).

    Materials and Methods:: Materials and Methods:  Hair soluble extracts (KTN) were prepared using reducing chemistry and capped with iodoacetamide (IAA).  Hydrophobic interaction chromatography (HIC) purification was employed using sodium phosphate buffer with linearly-decreasing salt gradient of ammonium chloride and a phenyl group (Hi Trap Phenyl HP) capture ligand.  For mass spectrometry (MS) identification, samples were first treated with dithiothreitol, urea, and IAA, trypsinized to break proteins into peptides, then ran in UPLC-MS: Waters Xevo G2-XS QTof using ESI+ (Mse) and leucine-enkephalin as the lockmass and visualized in MassLynx.  Data was processed in UNIFI using 16 hair keratin references (K31-39 (type I) and K81-86 (type II)) and Progenesis QI for proteomics (QIP, Waters).

    Results, Conclusions, and Discussions:: Results and Discussion:  The HIC process enabled successful separation and purification of type I and II keratin monomers (Fig. 1), observed via SDS-PAGE unique bands.  The KTN extract (Fig. 2) and HIC fractions showed good separation in UPLC-MS.  In UNIFI, the highest score and relative abundance in KTN were as follows: keratin-81, 32, 40, 38, 82, 35, 39, 84, 36, 33A, 31, 34, 37, 33B, 85, 83, and 86.  The higher MW-type II keratin eluate was found to be most likely K81 while the lower MW-type I determined to be K32.  Progenesis QIP verified the presence and identity of keratins in the KTN extract but also detected significant amounts of smaller-molecule globular matrix KAPs.  The purified HIC eluates were confirmed to be K81 and K32.

    Conclusion:  We developed a relatively quick keratin extraction and purification process using hydrophobic interaction liquid chromatography.  The purified hair keratins: K32 and K81, are currently being investigated for their assembly into heterodimers via transmission electron microscopy.  We are also developing a preparative scale HIC for purification and creation of scaffolds for tissue engineering use.


    Acknowledgements (Optional): : Acknowledgements:  Fusion Beauty Salon, Stylush Salon and Laser, Fresh Cuts, and Hair Designers for sources of human hair samples.  We also thank Rise Matsunami (Waters) for her guidance in proteomic analysis and technical support.

    References (Optional): :