Mapping Protein Cross-Links in Human Hair via Mass Spectrometry

Networks of protein-protein cross-links underpin all the key physicomechanical properties of mammalian fibers and also strongly influence how the fiber responds to treatments and environmental insults. During the last decade, significant improvement has been made in detecting and understanding cross-links, though, to date, mapping where these protein cross-links are within and between proteins has proved to be very challenging. This work reports a proof-of-concept study where mass spectrometry-based proteomics strategies were used to unravel the details of cross-link location between trichocyte keratin proteins in the hair shaft. This work focuses on two cross-links known to exist in hair proteins and that are used as a proxy for the degree of damage in hair proteins: lanthionine and lysinoalanine. Our results demonstrate that mass spectrometric evidence of the existence of both lanthionine and lysinoalanine cross-linked peptides within hair fibers can be found. The approach used also provided the first insight of the exact location of these cross-links within specific keratins. Analysis with respect to the current models of trichocyte intermediate filament organization indicates detected cross-links occur within antiparallel keratin heterodimers. The low abundance of the crosslinked peptides makes this type of evaluation approach difficult, but the results represent a new approach to untangle which cross-links are most essential to defining hair performance.