Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, USA; Department of Dermatology, University of Luebeck, Luebeck, Germany.
Proteomics and Metabolomics Facility, Wistar Institute, Philadelphia, Pennsylvania, USA.
J Invest Dermatol. 2018 Jun;138(6):1236-1242. doi: 10.1016/j.jid.2018.01.001.
Identifying previously unknown proteins or detecting the presence of known proteins in research samples is critical to many experiments conducted in life sciences, including dermatology. Sensitive protein detection can help elucidate new intervention targets and mechanisms of disease, such as in autoimmune blistering skin diseases, atopic eczema, or other conditions. Historically, peptides from highly purified single proteins were sequenced, with many limitations, by stepwise degradation from the N-terminus to the C-terminus with subsequent identification by UV absorbance spectroscopy of the released amino acids (i.e., Edman degradation). Recently, however, the availability of comprehensive protein databases from different species (derived from high-throughput next-generation sequencing of those organisms' genomes) and sophisticated bioinformatics analysis tools have facilitated the development and use of mass spectrometry for identification and global analysis of proteins, summarized as mass spectrometry-based proteomics. Mass spectrometry is an analytical technique measuring the mass (m)-to-charge (z) ratio of ionized biological molecules such as peptides. Proteins can be identified by correlating peptide-derived experimental mass spectrometry spectra with theoretical spectra predicted from protein databases. Here we briefly describe how this technique works, how it can be used for identification of proteins, and how this knowledge can be applied in elucidating human biology and disease.
鉴定研究样本中以前未知的蛋白质或检测已知蛋白质的存在,对生命科学领域的许多实验至关重要,包括皮肤病学。敏感的蛋白质检测可以帮助阐明新的干预靶点和疾病机制,如自身免疫性水疱性皮肤病、特应性皮炎或其他疾病。从历史上看,通过从 N 端到 C 端逐步降解,对高度纯化的单一蛋白质的肽进行测序,具有许多局限性,随后通过释放的氨基酸的紫外线吸收光谱(即 Edman 降解)进行鉴定。然而,最近,来自不同物种的综合蛋白质数据库(源自这些生物体基因组的高通量下一代测序)和复杂的生物信息学分析工具的可用性,促进了质谱用于蛋白质鉴定和全局分析的发展和使用,概括为基于质谱的蛋白质组学。质谱是一种分析技术,用于测量离子化生物分子(如肽)的质量(m)与电荷(z)比。通过将肽衍生的实验质谱谱与从蛋白质数据库预测的理论谱相关联,可以鉴定蛋白质。本文简要描述了该技术的工作原理、如何将其用于蛋白质鉴定,以及如何将这些知识应用于阐明人类生物学和疾病。