666 JOURNAL OF COSMETIC SCIENCE 1. First, though many cross-linked peptide ions can still not be detected due to the complexity of the sample, this study provides a first proof-of-principle that our approach, which targeted lanthionine and lysinoalanine cross-links, proved successful for mapping locations within human hair proteins. In each case, fragment ions that match both linked peptides were detected. 2. Second, the high homology in protein sequences in the keratin family complicates the identification of cross-linked proteins. We chose to report only the “first hit” protein (i.e., the protein with the highest score) during database matching. However, for some peptides it is possible that the peptide sequence of interest is not unique to just one protein. 3. Third, false positive cross-link identifications during data analysis require careful consideration. While our initial results mapped cross-links to both keratins and keratin- associated proteins, after applying stringent data analysis settings to minimize false positive identifications, in this work only linkages between keratins were confidently identified. POSITION OF THE CROSS-LINK WITHIN THE FIBER Next these preliminary mass spectrometric data were compared to the current models of trichocyte intermediate filament organization. Intermediate filament–forming keratin proteins can be classified into two major types: acidic type I keratins and neutral-basic type II keratins. Within the hair cortex, a type I and type II keratin form a coiled-coil heterodimer. To keep this three-dimensional coiled structure of the intermediate filament stable, protein–protein cross-links are crucial. Tetramers are formed when two of these heterodimers are clustered in an antiparallel fashion (9,11,12). Protofilaments cluster different tetramers, which end up in the intermediate filament structure (Figure 8). Three Figure 8. Schematic representation of the trichocyte keratin intermediate filament molecule. Each molecule is a heterodimer containing a type I and type II chain keratin. Tetramers are formed when two of these heterodimers are clustered in an antiparallel fashion. In these tetramers, three modes are common to all classes of intermediate filaments and involve (respectively) the approximate axial alignment of antiparallel 1B segments, antiparallel 2 segments, and antiparallel rod domains. Protofibrils cluster different tetramers, which end up in the intermediate filament structure.

667 MAPPING PROTEIN CROSS-LINKS IN HUMAN HAIR different modes of molecular assembly of intermediate filaments are described. These three modes are common to all classes of intermediate filaments and involve (respectively) the approximate axial alignment of antiparallel 1B segments (model A 11 ), antiparallel 2 segments (model A 22 ), and antiparallel rod domains (model A 12 ) (Figure 8). Previous work from the Parry et al. research groups (11,13–16) do show that newly assembled protofilament molecules associate in A 11 or A 22 alignments within the follicle before a profound structural rearrangement occurs when the environment changes from a reducing to an oxidizing one. We compared these filament models with our experimental data to get a better idea of what these positions of the cross-linked peptides within the protein sequence mean, and to see if our data aligned with the models of trichocyte intermediate filament organization. Important to note, though, is that this work is not intending to make any assumptions on the structural intermediate filament biology aspect. The lanthionine cross-link found between keratin 38 and keratin 85 matches the models in a number of ways: first, proteins keratin 38 and keratin 85 are, respectively, a type I and type II keratin and could thus form a heterodimer. They are both cortex proteins. Second, the position of the cross-linked peptides (Figure 3) demonstrates that the link is made between proteins in antiparallel positioned dimers forming a tetramer, with an interaction between a head domain of one protein and a tail domain of another. This could be an indication of the suggested model A 12 in a tetramer but could also be the packaging of adjacent tetramers. Third, the A 12 arrangement is probably the most prevalent in a mature hair fiber as it lends itself to optimal molecular packing and radial compaction in an oxidized environment (13). Subsequently, we researched whether the other detected lanthionine cross-links could also be explained by these models. Similar to the previous example, the cross-link between keratin 33A and keratin 74, detected in two spectra (Table I), did show an A 12 arrangement, with peptide 1 (AA 91–108) from keratin 33A located in linker 1, while peptide 2 from keratin 74 with amino acid position 370–378 in the sequence is part of coil 2. As these two proteins do not tend to be found in the same part of the fiber (i.e., K33a is in the cortex and medulla, while K74 is found in the inner root sheet), it is still a question of where this cross-link would be formed, as both peptides are unique and thus only identified in these respective proteins. The mass spectrometric evidence of the lanthionine cross-link between keratin 34 and keratin 85, a type I and type II keratin, does show a different positioning of the cross-linked peptides. Here, one peptide is found at the N-terminus (head region), while the other peptide is found in the linker 1 region (Figure 5). This lanthionine link would thus suggest that these two proteins are more comparable to the A 11 model, though the position of the head sequence in the intermediate filament is still questionable. Similarities between the proposed intermediate filament models are also found when looking at the trichocyte keratin lysinoalanine cross-links within the samples. Mass spectrometric mapping of a lysinoalanine cross-link between keratin 33B and keratin 80 displays an interesting link, as both proteins are present in the medulla. The medulla, however, is known to have a heterogenous structure, and hence, although the position of the peptides of interest shows that these are somewhat comparable to model A 22 , where the proteins are still present in antiparallel heterodimers within the filament, these may or may not form tetramers in the medulla. Though some mass spectrometric evidence for the presence of the other detected lysinoalanine cross-links is found, manual inspection of the data (e.g., the cross-link between keratin 34