Potential use of human hair shaft keratin peptide signatures to distinguish gender and ethnicity

Subject recruitment

A total of 40 healthy individuals of different genders and ethnicities but with the same age range (21–40 years) were recruited in the study in accordance to the ethical clearance granted by the Medical Ethics Committee of University of Malaya Medical Centre (MEC ID. NO: 20158-1577) (Institutional Review Board), which adheres to the Declaration of Helsinki. Informed written consent was acquired from all subjects prior to collection of their head hair samples. At least three strands of hair were collected from each subject and stored at −20 °C. In addition to visual inspection, information regarding subject’s hair treatment habits and relevant past medical histories, if any, were collected using a questionnaire. Relevant phenotypic characteristics of the hair shaft including discoloration, whitening, bleaching, weathering and perming, if any, were recorded. Subjects with previous history of bacterial or fungal-borne skin diseases, inflammation or cancer and/or under treatment for such ailments and those with chemically-treated hair were excluded from the study.

Isolation of human hair shaft proteins

Human hair shaft proteins were isolated using the alkaline solubilisation method in two stages as previously described (Wong et al., 2016; Budowle & Acton, 1981). Briefly, 5 mg of hair samples were sterilised with 90% ethanol, cut (1–4 mm) and incubated in 300 µl of lysis buffer at 90 °C for 30 min in the first stage. The resulting supernatant fluids were isolated using a QIAquick spin column (Qiagen, Hilden, Germany) and kept at 4 °C. The undissolved hair shaft fractions on the other hand, were transferred into a fresh tube containing 300 µl of lysis buffer with 0.1 M NaOH and pulverised by magnetic stirring for 30 min to increase the recovery of protein from the second stage of extraction. The resulting solutions containing hair shaft protein extract were centrifuged and the supernatant fluids were separated for subsequent precipitation of proteins using 4 °C acetone. Protein pellets were then solubilised in sample buffer and their concentrations were assayed using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) in accordance to the manufacturer’s guidelines.

2-dimensional electrophoresis and silver staining

Solubilised hair shaft proteins (100 µg) suspended in sample buffer were incubated in rehydration buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate, 2% immobilized pH gradient buffer pH 4-7, 0.002% w/w orange G and 7 mg of dithiothreitol (DTT) for 30 min at room temperature. The sample mixtures were rehydrated with 13 cm DryStrip gel pH 4-7 (GE Healthcare, Uppsala, Sweden) for 18 h at room temperature in a closed environment. The rehydrated strips were subjected to first dimensional gel electrophoresis separation using the Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare, Uppsala, Sweden) according to the following settings: (i) 500 V, 1 h, step and hold; (ii) 1,000 V, 1 h, gradient; (iii) 8,000 V, 2 h 30 min, gradient; (iv) 8,000 V, 55 min, step and hold. The focused strip was initially incubated with SDS equilibration buffer consisting of 6 M urea, 75 mM Tris–HCl, pH 8.8, 30% (v/v) glycerol, 2% sodium dodecyl sulphate (SDS) and 1% (w/v) DTT for 20 min, followed by a second equilibration in the same buffer but containing 4.5% (w/v) iodoacetamide instead of DTT for another 20 min. Second dimension separation was performed on 8–15% gradient polyacrylamide gel using the SE 600 Ruby electrophoresis system (GE Healthcare, Uppsala, Sweden). A concentration of 2.6% crosslinker (Bis) was used for the preparation of gels (% C). Cathode running buffer containing 0.025 M tris (pH 8.3), 0.192 M glycine and 0.1% SDS while, the anode buffer containing 0.375 M tris (pH 8.8) were used for the second dimensional gel electrophoresis separation. Four gels were run according to the following program: (i) 50 V, 150 mA, 100 W for 1 hr; (ii) 600 V, 150 mA, 100 W until tracker dye reached bottom of the gel. The electrophoresed gels were stained using silver nitrate (Yan et al., 2000). In this process, gels were firstly fixed with 40% (v/v) ethanol and 10% acetic acid for 30 min, followed by sensitization with 40% (v/v) ethanol, 0.5 M sodium acetate trihydrate and 8 mM sodium thiosulphate for 30 min. Gels were then washed thrice, each time for 5 min, and stained with 14.7 mM silver nitrate solution. After washing two more times for 1 min, gels were developed with 0.24 M sodium carbonate and 0.04% (v/v) formaldehyde. Development of spots was stopped with 40 mM EDTA solution and gels were finally kept in distilled water before being scanned (see next section). Subsequent to the present study, a report on modification of this 2-dimensional electrophoresis method, which generated further improved image resolution, was published (Wong, Hashim & Hayashi, 2019).

Data analysis

Silver-stained 2-dimensional electrophoresis gels were scanned using ImageScanner III (GE Healthcare, Uppsala, Sweden) and analysis of protein spot volume was performed using ImageMaster Platinum 7.0 software (GE Healthcare, Uppsala, Sweden). Image analysis was restricted to protein spot clusters that appeared consistently within each group of hair shaft proteins. The levels of proteins in each sample were calculated as a percentage of volume contribution (% vol) in which the volume of contribution refers to the volume percentage of a protein taken against the total spot volume of all the proteins, in order to eliminate the possible variations due to staining and/or protein loading. Automatic spot detection was performed with default parameters setting (cut-off parameters were Smooth-2; Saliency-1; Min area-5), whereas spot editing and removal of artefacts were done manually.

Data were analysed using the Statistical Package for Social Sciences (SPSS) version 25.0 (IBM Corporation, New York, USA). Shapiro–Wilk test of normality was used to assess the distribution of the datasets. Parametric Student’s t-test and ANOVA was used to analyse and identify significant changes in expression between two population means (gender) and several population groups (ethnicities), respectively, when the normality assumption was met (p > 0.05). Following ANOVA, a pairwise comparison procedure was performed to test all possible pairwise differences of the means using either Tukey HSD when equal variances was assumed or, Dunnett’s T3, if otherwise. On the other hand, non-parametric Mann–Whitney U and Kruskal–Wallis tests with rank-based post-hoc test was used as their respective counterparts when the assumption for normality was violated (p < 0.05). All values were presented as mean  ± 95% confidence interval, unless otherwise stated. A p value of less than 0.05 and fold change of more than 1.5-fold was considered significant.

Mass spectrometry and database search

Identification of proteins was performed as previously described with minor modifications (Seriramalu et al., 2010). Briefly, protein spots of interests were carefully cut out from 2-dimensional electrophoresis gels and kept in high-purity water at −20°C. Gel plugs were first destained using 15 mM potassium ferricyanide (III) and 50 mM sodium thiosulphate for 15 min at room temperature. The destaining procedure was repeated until the gel plugs became clear and transparent. The proteins in gel plugs were then reduced and alkylated using 10 mM DTT and 55 mM iodoacetamide both in 100 mM ammonium bicarbonate. They were then washed thrice with 50% acetonitrile in 100 mM ammonium bicarbonate, dehydrated with 100% acetonitrile and dried using vacuum centrifugation. The dried gels were treated with trypsin (6 µg/mL in 50 mM ammonium bicarbonate) for 18 h at 37 °C. The resulting peptides were then dried, reconstituted in formic acid (0.1%) and desalted using ZipTip with C18 resin (Millipore, Massachusetts, USA). The desalted and concentrated peptides were mixed with equal volume of α-cyano-4-hydroxycinnamic acid (6 mg/ml), before being spotted onto the OptiToF 384-well insert (0.7 µl) of the 5800 MALDI ToF/ToF analyser (SCIEX, Framingham, USA).

The proteins were identified using MASCOT search engine (Perkins et al., 1999) and the resulting mass spectral data were thoroughly queried against the human hair entries in the Uniprot database (last update: January 17, 2019, containing 1329 sequences). The following parameters were set: enzyme: trypsin; maximum missed cleavages: 1; fixed modification: carbamidomethylation of cysteine; variable modification oxidation of methionine; precursor ion mass tolerance: 100 ppm; fragment ion mass tolerance: 0.2 Da. An individual ion score of more than 17 indicates extensive homology or identity (p < 0.05).

Verification of altered protein abundance using immunoblotting

Immunoblotting was performed on pooled hair shaft protein samples of each groups of subjects, to verify the altered abundance of proteins observed in the 2-dimensional electrophoresis analysis. A total of 30 µg of proteins for each pooled sample, determined using Bio-Rad protein assay (Bio-Rad, California, USA), was separated on 12.5% SDS polyacrylamide gel and transferred onto PVDF membranes (0.2 µm, PALL Life Sciences, Port-Washington, USA) using a Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad, Hercules, USA) for 1 h at 100 V. Membranes were stained for total protein using MemCode™ Reversible Protein Stain Kit (Thermo Fisher Scientific, Waltham, USA), dried and scanned to be used as a loading control. After removal of Mem-Code™ staining, the membranes were blocked with 5% skim milk in 1 × TBST (25 mM Tris, 500 mM NaCl, 0.05% Tween, pH 7.5) for 30 min, washed thrice, 5 min with 1 × TBST, and incubated with anti-KRT86 antibody (ab192754) (Abcam, Cambridge, UK) overnight at 4 °C. After washing with 1 × TBST thrice for 5 min each wash, the membranes were incubated for 1 h with horseradish peroxidase conjugated goat anti-guinea pig IgG H&L antibody (ab6908) (Abcam, Cambridge, UK). The membranes were washed again thrice, 5 min with 1 × TBST and developed either using 3, 3′-diaminobenzidine chromogen solutions (Thermo Fisher Scientific, Waltham, USA) containing 0.02% hydrogen peroxide or WesternBright Sirius enhanced chemiluminescence system (Advansta, California, USA). MemCode™ signal and protein band intensities were obtained using the ImageJ Software (NIH, Bethesda, USA).

Hair shaft protein profiling—distinguishing genders and ethnicities

The extraction yields of protein (%) using the alkaline solubilisation method as previously described by Wong et al. (2016) were generally higher compared to the methods of Lee, Rice & Lee (2006) and Shindai (Fujii & Daguang, 2008). When alkaline solubilisation protein extraction method was applied to 5 mg of hair strands from 40 different individuals (30 females and 10 males), an average extraction yield of 63.56  ± 6.59 (mean  ± SD) was obtained.

When the human hair shaft protein extracts from female and male subjects of the Malay ethnic group were separated by 2-dimensional electrophoresis and subjected to silver staining, similar profiles were obtained (Figs. 1A and B). The gel profiles were then subjected to ImageMaster 2D Platinum Software and statistical analyses (see section: ‘Data Analysis’). Abundance of 6 hair shaft protein spots was found to be significantly higher in female compared to male subjects (Fig. 2). Spot 3 showed the highest mean percentage of volume contribution for females compared to males. The highest fold change difference was observed in spot 6 (Fig. 2).

When the same gel profiles of the female Malay subjects were compared to those generated from subjects of Chinese and Indian ethnicities of the same age range, 5 protein spots were significantly different in at least one ethnic group compared to the others (Fig. 3). Among the protein spots of altered abundance, spot 7 appeared the most intense. However, spot 11 demonstrated the highest fold change differences between Indian (0.244  ± 0.028) and Chinese (0.038  ± 0.079) subjects, as well as between Malay (0.147  ± 0.042) and Chinese (0.038  ± 0.027) subjects. When taken together, the analysis generally showed that the Indian female subjects had the highest mean percentage of volume contribution for spots 7, 9 and 11 compared to other ethnicities (females) whilst their spots 8 and 10 were the least intense. The Chinese subjects showed the highest mean percentage of volume contribution in spot 10 and lowest values for spots 9 and 11. In the Malay female subjects, spot 8 had the highest mean percentage of volume contribution, while spot 7 was the least intense.

Identification of hair shaft proteins by MALDI-ToF/ToF

The hair shaft protein spots of significant altered abundance between subjects of different genders and ethnicities were identified using MALDI-ToF/ToF analysis and search of the human hair database (Table 1). Analysis of the 11 hair shaft proteins of interest (Fig. 1) identified five different types of keratins, including (1) type II cuticular Hb6 (K86) for spots 3, 4, 5, 7 and 8, (2) type II cuticular Hb3 (K83) for spot 9, (3) type II cuticular Hb1 (K81) for spots 2, 6 and 10, (4) type II cuticular Hb5 (K85) for spot 1 and (5) type I cuticular Ha3-II (K33b) for spot 11. While the hair shaft of subjects of different genders demonstrated significant altered levels of K81, K85 and K86, those from different ethnicities showed significant different abundance of K33b, K81, K83 and K86.

Verification of K86 abundance

In this study, K86 was selected for verification of its abundances in the hair shaft of the subjects using immunoblotting method in view of its highest altered frequency as well as availability of commercialised antibodies against the protein. However, since the anti-K86 used is known to specifically recognise amino acids 446-469 of the keratin peptide, one of its peptide spots (spot 8), with amino acid sequences not in the region recognised by the antisera (Table S1), was not included in the analysis. Fig. 4 demonstrates the representative images generated from the immunoblotting experiments. The results obtained for K86 spots (3, 4, 5 and 7) appeared to corroborate the findings previously obtained via two-dimensional gel electrophoresis separation. K86 bands equivalent to the molecular weights of spots 3, 4 and 5 (spots 4 and 5 share similar molecular weight) appeared intensely stained in the female Malay subjects, relative to the male counterparts, while the female Indian subjects demonstrated more intense K86 bands compared to the female Malay and Chinese subjects. When analysed by densitometry, the intensity ratios for female Malay:male Malay bands for spots 3-, 4- and 5-equivalent K86 bands were <1.5-fold different, but significant (p < 0.05). On the other hand, the intensity ratios for the K86 spot 7-equivalent band for female Malay versus Indian subjects showed >1.5-fold difference (FC = 1.62, p = 0.008). The levels of spot 7-equivalent bands seemed comparable between the female Malay and Chinese (p > 0.05) subjects.