consumers with naturally curly hair have different cosmetic needs from people with straight hair (1), thus indicating that specifi c product design is required for hair-care products to deliver optimal benefi ts to each hair type. Hai r shape can be described by consumers as straight, wavy, curly, and a number of sub- jective variations thereof. An objective classifi cation system has been developed to classify these different hair shapes based on a number of morphological and phenotypical param- eters, independent of biogeographic or genomic ancestry. This classifi cation system, based on the degree of curliness, starts at type I which refers to straight hair and goes to type VIII which is tightly curly hair (2). Man y factors have been discovered over the years that have been claimed to directly or indirectly contribute to hair curvature. One of the most prominent fi ndings was that hair curvature correlates to the morphology of the hair follicle. It has been demonstrated that curly fi bers which emerge from the follicle are generated within curved follicles, whereas straight hair emerges from collinear follicles, with bulbs tilted at almost right angles with the scalp (3). Rotation of the follicle by the arrector pili muscle has been suggested as responsible for extrusion of helical fi ber shape (4). Others have suggested that the inner root sheath, which supports the growing hair shaft, plays a role in molding curvature due to correlations between mutations to keratins K71 and K74 and high-curl disorders (5). Challenging these extrusion views are studies that correlate internal hair-shaft features in the follicle or mature fi ber with hair shape such that follicle shape is a consequence of fi ber development and not vice versa. In the follicle, these include reported differences across the hair shaft in cell division rates (6) and keratin structure development (7,8). In the mature hair, an elliptical profi le is often cited as correlated to curliness (5,9), but there are other differences across the hair shaft at the microstructural level in keratin nanostructure organization (10–12) and cortical cell remnant length (13) that also correlate with hair curvature. Two main points are that the complexity of hair growth makes discriminating between cause and effect diffi cult (14,15) and that hair curliness appears to be pro- grammed within the follicle bulb (16,17). Dat a that clarify the underpinning mechanism that connects the proteins and microstruc- ture with curvature in human hair will help us not only understand hair shape generation but more directly identify differences in damage and interventions related to hair shape. In this study, we target a comprehensive understanding of the differences in hair curva- ture at the molecular level by applying proteomic technologies to decipher differences in the proteome between two groups with distinct hair shapes. The fi rst group refl ects hair samples with very straight hair, whereas the second group contains hair samples with very high levels of curvature. MET HODS OVE RVIEW OF SAMPLES Hai r fi ber samples were sourced from 50 subjects with very straight hair, with character- istics of type I in the scale defi ned by Loussouarn and de la Mettrie et al. (18), and from 50 panelists with very curly hair, with characteristics of types V–VIII of the same scale. All subjects were aged between 18 and 55 years and had not chemically treated their hair for at least 6 weeks before sampling. JOURNAL OF COSMETIC SCIENCE 250
Pre screening was performed to identify a large number of subjects who may meet the required study criteria by review of expert assessments of hair type from the databases of the Unilever Consumer Science Centre, Unilever R&D, Port Sunlight, UK, and of a con- sumer research partner. Subjects identifi ed with the required hair types were then contacted to communicate the study objectives and criteria and the hair sampling requirements subjects meeting the study criteria and willing to participate were then invited to a study center for a further assessment. At the study center, an expert confi rmed the subject hair type met the study recruitment criteria and the absence of any hair or scalp medical con- ditions before fi ber sampling. To sample hair fi bers with minimal exposures to cosmetic treatments, or cosmetic inter- ventions, the hair was cut by a hairdresser very close to the scalp from four locations on the head. Before sampling, the subjects’ hair style was parted in the center and then again, normal to the fi rst, to create four quadrants. Each quadrant was then parted hori- zontally approximately 2 cm above eye level for sampling. Approximately 50 hair fi bers were cut close to each quadrant’s parting along a narrow line to minimize the cosmetic impact of the hair removal. Cut samples from each region of the head were then bundled and secured in a device with the root cut end clearly identifi ed for further processing. SAM PLE PREPARATION Bef ore proteomic analysis, all the hair samples were washed in 1.4% sodium laurel sulfate with gentle agitation and rinsed twice with water. Samples were air-dried and the root-most ~2 cm was cut off and weighed. To minimize biological variation, 20 pools (A–J: very curly hair and K–T: very straight hair) were created with similar weights of hair (±10%) from fi ve different individuals in each pool. After pooling, the samples were snipped to approximately 1-mm lengths and stored in airtight glass containers protected from light. The snipped hair samples were further rinsed 10 times with liquid-chromatography mass spectrometry (LCMS)-grade water and air-dried before mass spectrometric analysis. PROT EOMIC ANALYSIS Samp le preparation Prot eins from 10 mg of each fi ne snipped subsample were extracted with 1 mL of extraction buffer containing 7 M urea, 2 M thiourea, 50 mM dithiothreitol, 50 mM tris(hydroxymethyl) aminomethane, 5% sodium deoxycholate, and pH 7.5. After 18 hours of vigorous shaking at 35°C in an Eppendorf thermomixer (Merck, Kenilworth, NJ), the solubilized proteins were precipitated with a chloroform/methanol procedure. After removal of methanol and air-drying, the protein pellet was resuspended in 100 μL 0.01 M ammonium bicarbonate. The protein concentrations of the hair extracts were quantifi ed using a Direct Detect in- strument (Merck) according to the manufacturer’s instructions, and a volume equivalent to 150 μg of protein from each sample was dried in a vacuum centrifuge. The dried protein was resuspended in 0.1 M ammonium bicarbonate and chemically reduced by agitation with 20 μL of 50 mM dithiothreitol at 56°C for 45 minutes. The proteins were then alkylated by the addition of 20 μL of 150 mM iodoacetamide, with agitation at room temperature in the dark for 30 minutes. Sequencing grade trypsin (3 μg) was HAIR SHAPE PROTEOMICS 251
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