668 JOURNAL OF COSMETIC SCIENCE and keratin 82 [Figure 7]) shows that the peptides are detected with fragments of very low intensity, and a distinction between the fragment ions and the background signals is hard to detect. Therefore, none of the other lysinoalanine data were taken forward for further comparison against the intermediate filament models. In conclusion, our results demonstrate here, for the first time, the mass spectrometric characterization of native cross-links within hair fibers (lanthionine and lysinoalanine), combined with their mapping within the protein sequence, and their potential mapping within the intermediate filament. These results represent the first steps toward untangling the many unknowns related to cross-links in fibers. However, caution should still be taken, as our results clearly indicate that the low abundance of the cross-linked peptides still makes this type of evaluation approach difficult, even when optimized mass spectrometric data acquisition methods are applied. These preliminary data, however, do show evidence of previous reported heterodimer compositions and do showcase the potential of the technology. ACKNOWLEDGMENTS We would like to thank Dr. Duane Harland and Dr. Jeffrey Plowman for their critical input in this work. This work was partially funded and supported by a contract with the Procter & Gamble Company and by the AgResearch Strategic Science Investment Fund. REFERENCES (1) C. R. Robbins, “Morphological, macromolecular structure and hair growth,” Chemical and Physical Behavior of Human Hair (Springer, Berlin, Heidelberg, 2012), pp. 1–104. (2) D. P. Harland, R. J. Walls, J. A. Vernon, J. M. Dyer, J. L. Woods, and F. Bell, Three-dimensional architecture of macrofibrils in the human scalp hair cortex, J. Struct. Biol., 185(3), 397–404 (2014). (3) S. Deb-Choudhury, Crosslinking between trichocyte keratins and keratin-associated proteins, Adv. Exp. Med. Biol., 1054, 173–183 (2018). (4) A. L. Miranda-Vilela, A. J. Botelho, and L. A. Muehlmann, An overview of chemical straightening of human hair: technical aspects, potential risks to hair fibre and health and legal issues, Int. J. Cosmet. Sci., 36(1), 2–11 (2014). (5) E. Maes, J. M. Dyer, H. J. McKerchar, S. Deb-Choudhury, and S. Clerens, Protein–protein cross-linking and human health: the challenge of elucidating with mass spectrometry, Expert Rev. Proteomics, 14(10), 917–929 (2017). (6) H. J. McKerchar, S. Clerens, R. C. J. Dobson, J. M. Dyer, E. Maes, and J. A. Gerrard, Protein–protein crosslinking in food: proteomic characterisation methods, consequences and applications, Trends in Food Science & Technology, 86, 217–229 (2019). (7) M. R. Hoopmann, A. Zelter, R. S. Johnson, M. Riffle, M. J. MacCoss, T. N. Davis, and R. L. Moritz, Kojak: efficient analysis of chemically cross-linked protein complexes, J. Proteome Res., 14(5), 2190–2198 (2015). (8) L. Kall, J. D. Canterbury, J. Weston, W. S. Noble, and M. J. MacCoss, Semi-supervised learning for peptide identification from shotgun proteomics datasets, Nat. Methods, 4(11), 923–925 (2007). (9) R. D. B. Fraser and D. A. D. Parry, Structural hierarchy of trichocyte keratin intermediate filaments, Adv. Exp. Med. Biol., 1054, 57–70 (2018). (10) E. Maes, S. Clerens, J. M. Dyer, and S. Deb-Choudhury, Improved detection and fragmentation of disulphide-linked peptides, Methods Protoc., 1(3), (2018).

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