654 JOURNAL OF COSMETIC SCIENCE (67) D. P. Harland and J. E. Plowman, “Development of hair fibres,” in The Hair Fibre: Proteins, Structure and Development. J. E. Plowman, D. P. Harland, and S. Deb Choudhury. Eds. (Springer, New York, NY, 2018), pp. 109–154. (68) L. Auber, The anatomy of follicles producing wool-fibres, with special reference to keratinization, Trans. R. Soc. Edinb., 62(1), 191–254 (1952). (69) R. E. Chapman, “The ovine arrector pili musculature and crimp formation in wool,” in Biology of the Skin and Hair Growth. A. G. Lyne and B. F. Short. Eds. (Angus and Robertson, Sydney, Australia, 1965), pp. 201–232. (70) J. J. Lemasters, V. K. Ramshesh, G. L. Lovelace, J. Lim, G. D. Wright, D. Harland, and T. L. Dawson, Compartmentation of mitochondrial and oxidative metabolism in growing hair follicles: a ring of fire, J. Invest. Dermatol., 137, 1434–1444 (2017). (71) A. J. Mickinnon, D. P. Harland, and J. L. Woods, Relating self-assembly to spatio-temporal keratin expression in the wool follicle, J. Text. Eng., 62, 123–128 (2016). (72) D. F. Orwin and J. L. Woods, Number changes and development potential of wool follicle cells in the early stages of fiber differentiation, J. Ultrastruct. Res., 80, 312–322 (1982). (73) M. S. Zamil, D. P. Harland, B. K. Fisher, M. G. Davis, J. R. Schwartz, and A. Geitmann, Biomechanics of hair fibre growth: a multi-scale modeling approach, J. Mech. Phys. Solids, 148, 104290 (2021). (74) Z. Yu, S. W. Gordon, A. J. Nixon, C. S. Bawden, M. A. Rogers, J. E. Wildermoth, N. J. Maqbool, and A. J. Pearson, Expression patterns of keratin intermediate filament and keratin associated protein genes in wool follicles, Differentiation, 77(3), 307–316 (2009). (75) J. McKinnon and D. P. Harland, A concerted polymerization-mesophase separation model for formation of trichocyte intermediate filaments and macrofibril templates 1: Relating phase separation to structural development, J. Struct. Biol., 173(2), 229–240 (2011). (76) P. I. Hynd, N. M. Edwards, M. Hebart, M. McDowall, and S. Clark, Wool fibre crimp is determined by mitotic asymmetry and position of final keratinisation and not ortho- and para-cortical cell segmentation, Animal, 3, 838–843 (2009). (77) E. Maes, F. Bell, C. Hefer, A. Thomas, D. Harland, A. Noble, J. Plowman, S. Clerens, and A. Grosvenor, Insights in human hair curvature by proteome analysis of two distinct hair shapes, J. Cosmet. Sci., 72(3), 249–267 (2021). (78) J. E. Plowman, R. E. Miller, A. Thomas, A. J. Grosvenor, D. P. Harland, and S. Deb-Choudhury, A detailed mapping of the readily accessible disulfide bonds in the cortex of wool fibers, Proteins, 89(6), 708–720 (2021). (79) E. Maes, J. M. Dyer, S. Deb-Choudhury, S. Clerens. Mapping protein crosslinks in human hair via mass spectrometry. J. Cosmet. Sci., 72(S), 655–669 (2021). (80) J. E. Plowman, D. P. Harland, S. Ganeshan, J. L. Woods, B. van Shaijik, S. Deb-Choudhury, A. Thomas, S. Clerens, and D. R. Scobie, The proteomics of wool fibre morphogenesis, J. Struct. Biol., 191(3), 341–351 (2015). (81) L. Langbein, M. A. Rogers, H. Winter, S. Praetzel, U. Beckhaus, H.-R. Rackwitz, and J. Schweizer, The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle, J. Biol. Chem., 274, 19874–19884 (1999). (82) L. Langbein, M. A. Rogers, H. Winter, S. Praetzel, and J. Schweizer, The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins, J. Biol. Chem., 276, 35123–35132 (2001). (83) L. Langbein and J. Schweizer, Keratins of the human hair follicle, Int. Rev. Cytol., 243, 1–78 (2005). (84) L. Langbein, H. Yoshida, S. Praetzel-Wunder, D. A. Parry, and J. Schweizer, The keratins of the human beard hair medulla: the riddle in the middle, J. Invest. Dermatol., 130, 55–73 (2010).

655 Address all correspondence to Evelyne Maes, evelyne.maes@agresearch.co.nz Mapping Protein Cross-Links in Human Hair via Mass Spectrometry EVELYNE MAES, JOLON M. DYER, SANTANU DEB-CHOUDHURY AND STEFAN CLERENS Lincoln Research Centre, AgResearch Ltd, Christchurch, New Zealand (E.M., J.M.D., S.D.-C., S.C.) Riddet Institute, Massey University, Palmerston North, New Zealand (E.M., J.M.D., S.C.) Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand (E.M., J.M.D., S.C.) Synopsis 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 cross- linked 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. INTRODUCTION Human hair is a complex tissue with fibers consisting of three morphological compartments composed of cornified dead cells: the cuticle, the cortex, and the medulla. The medulla is a loosely packed region in the center of the hair shaft, surrounded by the cortex, which represents the major structure in hair. To protect the cortex against the outside environment, a layer of overlapping cells called the cuticle surrounds the cortex (1). The hair cortex is primarily composed of proteins (90–95%), with lipids, water, and trace elements as the other chemical constituents present. The cortical proteome is dominated by two classes of proteins: first, intermediate filament–forming trichocyte (or alpha) keratins, and second, keratin-associated proteins that surround the keratin filaments. The keratins associate J. Cosmet. Sci., 72, 655–669 (November/December 2021)