Kiatthitinan Phonchawit, Yee Darian H, Tran Huu M Q, Sun Bingyun
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Rapid Commun Mass Spectrom. 2025 Sep 15;39(17):e10071. doi: 10.1002/rcm.10071.
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.
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.
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 values exceeding 0.95 in pairwise quantitative comparisons via spectra counting across different operators, processes, and laboratories.
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.
毛发杆部的蛋白质组已越来越多地通过质谱法进行研究，以便对主要毛发蛋白质（如用于生物医学和法医应用的表皮角蛋白）进行灵敏、准确且全面的表征。作为人类皮肤的外部附属器官，头皮毛发杆部由生物学和化学性质稳定的死亡角质化细胞构成。毛发杆部蛋白质的提取和消化一直是毛发蛋白质组学的瓶颈。
我们介绍了一种在温和碱性条件下使用商用Precellys匀浆器的简单可靠的样品制备程序。我们通过进行过夜胰蛋白酶消化以实现部分蛋白水解，而非3天的完全消化，进一步缩短了样品制备程序。
使用该方法，我们从人类头皮毛发杆部实现了超过75%的蛋白质提取效率，且有限的蛋白水解提高了角蛋白序列覆盖率。我们方法的稳健性通过高重现性得到证实，在不同操作人员、流程和实验室之间通过光谱计数进行的成对定量比较中，R值超过0.95。
我们开发了一种用于人类毛发杆部的简便且稳健的样品制备策略。通过缩短和不完全的蛋白水解提高表皮角蛋白的序列覆盖率对于鉴定角蛋白中的基因变异肽至关重要。
