659 MAPPING PROTEIN CROSS-LINKS IN HUMAN HAIR LOCATION OF LANTHIONINE CROSS-LINKS WITHIN HAIR PROTEIN EXTRACTS VIA MASS SPECTROMETRY As cross-linked peptides are very challenging to characterize by mass spectrometry, a data acquisition method that allows for better fragmentation of these low-abundant cross-linked peptide precursors was developed. By allowing a more dynamic sampling of precursors in the mass spectrometer, a higher chance of detecting low-abundant, multiple-charged precursors was created. To maximize the chance of detecting lanthionine and lysinoalanine cross-links between keratin-related proteins and to minimize the amount of false positive hits within this process, we designed a custom database that consisted of all human keratins and keratin-associated proteins, as it is believed that cross-links formed within these protein families are largely responsible for the hair fiber rigid structure. Using this FASTA file (2019.02), we analyzed for cross-links between 298 proteins (including decoy proteins), resulting in 22,204 in silico tryptic peptide combination possibilities, of which 8,759 peptides are linkable. Table I lists lanthionine cross-links that could be detected in the hair digests. The table lists the two potential peptide sequences that are linked by a lanthionine cross-link as well as their corresponding first hit proteins. It also provides the spectrum number so that further manual validation of the fragmentation spectrum is possible. The spectrum-match validation settings were chosen as very stringent, and only q values smaller than 0.05 were considered confident. Because the algorithm only reports q values to three decimal places, the q values that made it through the filter are reported as 0. A further look at the fragmentation spectrum of the detected cross-link is depicted in Figure 2. A few observations can be made. First, as lanthionine derives from dehydroalanine intermediates (6), the bond can exist between cysteine–cysteine or serine–cysteine in the cross-linked peptides. All the cross-linked peptides detected consisted of a cysteine– cysteine bond. Second, none of the annotated fragment ions are ions that are directly linked to the Cys–Cys bond, though several other ions are detected. Third, the detected fragment ions are still all very low abundance, yet through our dynamic acquisition method, just detectable. Fourth, the detected b-ions and y-ions originating from the two peptides, the dominant alpha peptide, as well as the beta peptide are indicated. Table I List of Lanthionine Cross-Links Found in Human Virgin Hair Shaft Digest Scan number q value Peptide Peptide Protein 1 Protein 2 19,599 0 .LLEGEEHRLC[57.02]​ EGVGSVN​ VCVSSSRGGVSC​ [57.02]GGLSYSTTPGR NVSVSPIDIGC[57.02] QPGAEAN​ IAPM[15.99] CLLANVAHAN​ [0.98]R HUMAN_K85 HUMAN_K38 6,471 0 .QVVSSSEQLQ[0.98] SCQAEIIELR ISSGCGVTRNFSS​ CSAVAPK HUMAN_K34 HUMAN_K85 4,490 0 .SQQQEPLVCASYQSYFK CEIGNVK HUMAN_K33A HUMAN_K74 4,479 0 .SQQQEPLVCASYQSYFK CEIGNVK HUMAN_K33A HUMAN_K74 3,540 0 .SQQQEPLLCPSYQSYFK MSCRSYR HUMAN_K34 HUMAN_K84

Figure 2. Mass spectrometric evidence of the existence of lanthionine inter–cross-links between two peptides from keratin 38 and keratin 85. The data visualize scan number 19,599 of Table I. 660 JOURNAL OF COSMETIC SCIENCE