Studies on the Proteome of Human Hair - Identification of Histones and Deamidated Keratins

Abstract

Human hair is laminar-fibrous tissue and an evolutionarily old keratinization product of follicle trichocytes. Studies on the hair proteome can give new insights into hair function and lead to the development of novel biomarkers for hair in health and disease. Human hair proteins were extracted by detergent and detergent-free techniques. We adopted a shotgun proteomics approach, which demonstrated a large extractability and variety of hair proteins after detergent extraction. We found an enrichment of keratin, keratin-associated proteins (KAPs), and intermediate filament proteins, which were part of protein networks associated with response to stress, innate immunity, epidermis development, and the hair cycle. Our analysis also revealed a significant deamidation of keratin type I and II, and KAPs. The hair shafts were found to contain several types of histones, which are well known to exert antimicrobial activity. Analysis of the hair proteome, particularly its composition, protein abundances, deamidated hair proteins, and modification sites, may offer a novel approach to explore potential biomarkers of hair health quality, hair diseases, and aging.

Figure 1

Human hair proteins identified by LC-MS/MS and their physiochemical properties. (a) Number of proteins identified in urea, SDSI and SDSII hair extracts by LC-MS/MS. (b) Statistical analysis (ANOVA) of proteins identified by LC-MS/MS. (c) The molecular weights and pI values of LC-MS/MS identified proteins are presented.

Figure 2

Scanning electron microscope (SEM) images of hair shaft showing surface morphology before and after protein extraction techniques. (a) Surface morphology of unextracted hair (Control). (b) Hair after one time of SDS extraction (SDSI). (c) Hair after two SDS extractions (SDSII). (d) Hair after urea extraction.

Figure 3

Hierarchical clustering of keratin and keratin-associated proteins identified in the “extractome” of hair using different extraction methods. Highly abundant protein values are displayed in red, low abundance is indicated by blue, and intermediate values are in different shades of red and blue.

Figure 4

Mass-spectrometry-based proteomic identification and western blot detection of histones, their abundances determined by protein score and emPAI values. (a) Table showing different histones identified by mass spectrometry. (b) Western blot analysis illustrating presence of different histones (full lengths WBs of histones are provided in supplementary information Figs S4–S6). (c) Abundances of different histones.

Figure 5

Venn diagram showing overlap of Unique N- and Q-deamidation peptides and modification sites identified in hair proteome extracted by different techniques. Overlap analysis of (a) Unique Q-deamidated peptides, (b) Unique Q-modified sites, (c) Unique N-deamidated peptides, and (d) Unique N-deamidated sites detected using the different extraction techniques (Urea, SDSI and SDSII).