JOURNAL OF COSMETIC SCIENCE 234 INTERPRETATION AND PROSPECTS The nail unit and hair follicle produce structures that are remarkably tough and cohesive by virtue of their protein components, an abundance of intramolecular and intermolecular disul- fi de bonds and translgutaminase-mediated isopeptide bonding connecting the intermediate fi lament matrix to the cell surface. The fi nal stage of the intricate keratinocyte differentiation program involves transglutaminase activation by increasing concentration of intracellular cal- cium ions, thereby connecting membrane and junctional proteins to the cytoskeleton. The enzymes encoded by the TGM1 and TGM3 genes appear effective in that most of the proteins found in the solubilized fraction are also found in the insoluble (cross-linked) fraction of both hair shaft and nail plate. That so many proteins from all the compartments of the cell were identifi ed in the insoluble fraction is evidence that the corneocyte incorporates available pro- tein rather than a few specifi c proteins. A future goal for hair is to analyze the proteome of cuticle, cortex and medulla cells separately. This has now been accomplished for wool cuticle, where 100 proteins were identifi ed representing a variety of cellular processes (27). Hair shaft, nail plate and epidermal callus, the latter through sampling with tape strips, provide essentially noninvasive sources of discrete protein subsets of the total organismal proteome. The shotgun approach to analysis is even anticipated to permit distinguishing the proteomes of epidermis at various anatomic sites, including glabrous surfaces or those infl uenced by adjoining abnormal conditions (e.g., acne). For analysis of disease states, it has the advantage of surveying many gene products in parallel, permitting discovery of single components that may be defi cient. While homozygous protein loss may be readily detect- able, the approach does have obvious limitations for identifying the basis for any given disease or adverse condition, since only the most prominent proteins are surveyed. Even if a given protein is identifi ed in the sample examined, a point mutation could easily be over- looked if the protein coverage did not include the peptide in which the mutation occurs, or the affected peptide, one of many unique peptides, is not specifi cally monitored. Neverthe- less, downstream effects of a given defect might still be visible in altered levels of other proteins that may be important for the phenotype. Detection of a heterozygous defective allele in a carrier of a recessive condition is also problematic without better quantitation. Complementing the above shotgun (or discovery) approach, targeting specifi c peptides has potential utility. For example, quantitating only a small number of proteotypic peptides from a given protein, those unique to that protein and obtained reproducibly in high yield with suitable fragmentation patterns, can provide improved relative quantitation (28). This approach (multiple reaction monitoring) would be attractive if it would permit de- tection of heterozygous gene loss. For those diseases where a limited number of mutations were anticipated, and they occurred in suitable locations in a detectable protein, expected mutant peptides could be monitored. Additionally, focusing on specifi c peptides has the advantage of permitting much greater sensitivity. Ultimately, a desirable outcome would be to use a panel of specifi c peptides to distinguish alternative diagnoses for given diseases or conditions. While this would not replace the gold standard of genetic testing, it could serve as a useful screen in a comprehensive diagnostic paradigm. ACKNOWLEDGMENTS This work was supported by USPHS grant P42 ES04699 from the National Institute of Environmental Health Sciences. I thank Drs. Young Jin Lee, Rich Eigenheer and Brett

2010 TRI/PRINCETON CONFERENCE 235 Phinney of the Proteomics Core, University of California, Davis for mass spectrometry guidance and data collection and Dr. Ai Hayashi for expert technical assistance with mea- suring hair shaft diameters. REFERENCES (1) G. Matoltsy and C. A. Balsamo, A study of the components of the cornifi ed epithelium of human skin, J. Biophys. Biochem. Cytol., 1, 339–361 (1955). (2) A. G. Matoltsy, “The Membrane of Horny Cells,” in Biochemistry of Cutaneous Epidermal Differentiation, M. Seiji and I.A. Bernstein, Eds. (University of Tokyo Press, 1977), pp. 93–109. (3) R. H. Rice, M. Mehrpouyan, Q. Qin, and M. A. Phillips, “Transglutaminases in Keratinocytes,” in The Keratinocyte Handbook, I.M. Leigh, B. Lane, and F.M. Watt, Eds. (Cambridge University Press, 1994), pp. 259–274. (4) R. H. Rice, D. M. Rocke, H.-S. Tsai, Y. J. Lee, K. A. Silva, and J. P. Sundberg, Distinguishing mouse strains by proteomic analysis of pelage hair, J. Invest. Dermatol., 129, 2120–2125 (2009). (5) R. H. Rice, V. J. Wong, and K. E. Pinkerton, Ultrastructural visualization of cross-linked protein fea- tures in epidermal appendages, J. Cell Sci., 107, l985–1992 (1994). (6) R. H. Rice, D. Crumrine, D. Hohl, C. S. Munro, and P. M. Elias, Cross-linked envelopes in nail plate in lamellar ichthyosis. Br. J. Dermatol., 149, 1050–1054 (2003). (7) R. H. Rice, D. Crumrine, Y. Uchida, R. Gruber, and P. M. Elias, Structural changes in epidermal scale and appendages as indicators of defective TGM1 activity, Arch. Dermatol. Res., 297, 127–133 (2005). (8) R. H. Rice, V. J. Wong, V. H. Price, D. Hohl, and K. E. Pinkerton, Cuticle cell defects in lamellar ichthyosis hair and anomalous hair shaft syndromes visualized after detergent extraction, Anatomic Rec., 246, 433–440 (1996). (9) R. H. Rice, V. J. Wong, M. L. Williams, V. H. Price, D. Hohl, J. P. Sundberg, et al., Hair shaft defects visualized after detergent extraction, Exp. Dermatol., 8, 308–310 (1999). (10) R. H. Rice, V. J. Wong, K. E. Pinkerton, and J. P. Sundberg, Cross-linked features of mouse pelage hair resistant to detergent extraction, Anatomic Rec., 254, 231–237 (1999). (11) S. I. Chung and J. E. Folk, Transglutaminase from hair follicle of guinea pig. Proc. Natl. Acad. Sci. USA, 69, 303–307 (1972). (12) H. W. J. Harding and G. E. Rogers, Formation of the ε-(γ-glutamyl) lysine cross-link in hair proteins. In- vestigation of transamidases in hair follicles, Biochemistry, 11, 2858–2863 (1972). (13) L. Lorand, Fibrinoligase: The fi brin-stabilizing factor system of blood plasma, Ann. NY Acad. Sci., 202, 6–30 (1972). (14) T.-T. Sun and H. Green, Differentiation of the epidermal keratinocyte in cell culture: Formation of the cornifi ed envelope, Cell, 9, 511–521 (1976). (15) S. M. Thacher and R. H. Rice, Keratinocyte-specifi c transglutaminase of cultured human epidermal cells: Relation to cross-linked envelope formation and terminal differentiation, Cell, 40, 685–695 (1985). (16) S. Thibaut, N. Cavusoglu, E. de Becker, F. Zerbib, A. Bednarczyk, C. Schaeffer, et al., Transglutaminase-3 enzyme: A putative actor in human hair shaft scaffolding? J. Invest. Dermatol., 129, 449–459 (2009). (17) R. H. Rice, G. E. Means, and W. D. Brown, Stabilization of bovine trypsin by reductive methylation, Biochim. Biophys. Acta, 492, 316–321 (1977). (18) B. C. Searle, Scaffold: A bioinformatic tool for validating MS/MS-based proteomic studies, Proteomics, 10, 1265–1269 (2010). (19) Y. J. Lee, R. H. Rice, and Y. M. Lee, Proteome analysis of human hair shaft: From protein identifi cation to posttranslational modifi cation, Molec. Cell. Proteom., 5, 789–800 (2006). (20) R. H. Rice and H. Green, The cornifi ed envelope of terminally differentiated human epidermal kerati- nocytes consists of cross-linked protein, Cell, 11, 417–422 (1977). (21) Y. Ishihama, Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber, et al., Exponentially modifi ed protein abundance index (emPAl) for estimation of absolute protein amount in proteomics by the number of se- quenced peptides per protein, Molec. Cell. Proteom., 4, 1265–1272 (2005). (22) C. Seibert, B. R. Davidson, B. Fuller, L. H. Patterson, W. J. Griffi ths, and Y. Wang, Multiple approaches to the identifi cation and quantifi cation of cytochromes P450 in human liver tissue by mass spectrometry, J. Proteome Res., 8, 1672–1681 (2009).