Abstract
RATIONALE: The proteome of the hair shaft has been increasingly studied by mass spectrometry for sensitive, accurate, and comprehensive characterization of major hair proteins such as cuticular keratins for biomedical and forensic applications. As an external appendage of human skin, the shaft of scalp hair is formed by dead keratinized cells that are biologically and chemically stable. The extraction and digestion of hair shaft proteins have been bottlenecks in hair proteomics. METHODS: We present a straightforward and reliable sample preparation procedure using a commercial Precellys homogenizer in mild basic conditions. We further shortened the sample preparation procedure by implementing an overnight tryptic digestion for partial proteolysis instead of a 3-day complete digestion. RESULTS: Using this method, we achieved over 75% protein extraction efficiency from the shaft of human scalp hair, and the limited proteolysis improved keratin sequence coverage. The robustness of our method was confirmed by high reproducibility, with R(2) values exceeding 0.95 in pairwise quantitative comparisons via spectra counting across different operators, processes, and laboratories. CONCLUSIONS: We developed a facile and robust sample preparation strategy for human hair shafts. The improved sequence coverage in cuticular keratins by shortened and incomplete proteolysis is critical for the identification of genetically variant peptides in keratins.