Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.
Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Campus Kiel, Germany.
J Proteomics. 2020 Apr 15;217:103678. doi: 10.1016/j.jprot.2020.103678. Epub 2020 Feb 6.
Analysis of the human skin proteome is key to understand molecular mechanisms maintaining health or leading to diseases of this important organ. For minimal invasive sampling of skin proteomes, the use of self-adhesive tape strips has been successfully applied. However, the methods previously presented were evaluated on different types of skin samples (e.g. healthy, diseased) and used a variety of cell lysis/protein extraction methods, which renders a systematic comparison and thus the identification of the most efficient protocols difficult. Here, we present a study comparing five different approaches for cell lysis and protein extraction from single tape strip biopsies. Extraction using a detergent mix or 1% SDS proved to be most efficient. Further, we replaced protein precipitation by single-pot, solid-phase-enhanced sample preparation (SP3), which strongly enhanced the number of identified proteins. This fully LC-MS compatible methodology provides a fast and reproducible approach for minimal invasive sampling of human skin proteomes. BIOLOGICAL SIGNIFICANCE: Fast and reproducible minimal invasive sampling of human skin proteomes is a major prerequisite for clinical proteomics studies aiming to decipher molecular mechanisms involved in the homeostasis as well as in the development of diseases. By optimization of tape strip sampling, e.g. the introduction of SP3 sample cleanup prior to LC-MS analysis, the presented protocol leads to yet not reported numbers of protein identifications from healthy human skin. Further, due to its efficiency it allows analysis from minimal sample amounts, e.g. from single tape strips, while established protocols relied on pooling of multiple tape strips. This provides the opportunity to perform spatially (lateral) resolved proteome analyses from different depths of the skin by analysis of consecutive strips.
分析人类皮肤蛋白质组对于理解维持健康或导致该重要器官疾病的分子机制至关重要。为了对皮肤蛋白质组进行微创采样，已经成功应用了自粘胶带条。然而，以前提出的方法是在不同类型的皮肤样本（例如健康、患病）上进行评估的，并且使用了各种细胞裂解/蛋白质提取方法，这使得系统比较和因此确定最有效的方案变得困难。在这里，我们比较了从单个胶带条活检中进行细胞裂解和蛋白质提取的五种不同方法。使用去污剂混合物或 1%SDS 进行提取被证明是最有效的。此外，我们通过单锅固相增强样品制备（SP3）代替蛋白质沉淀，这大大增强了鉴定蛋白质的数量。这种完全与 LC-MS 兼容的方法为微创采样人类皮肤蛋白质组提供了一种快速且可重复的方法。生物学意义：快速且可重复的微创采样人类皮肤蛋白质组是临床蛋白质组学研究的主要前提，旨在破译涉及内稳定以及疾病发展的分子机制。通过优化胶带条采样，例如在 LC-MS 分析之前引入 SP3 样品净化，可以从健康的人类皮肤中获得尚未报道的蛋白质鉴定数量。此外，由于其效率高，它允许从小量样本进行分析，例如单个胶带条，而既定的方案依赖于多个胶带条的组合。这为通过分析连续的条带来实现皮肤不同深度的空间（横向）分辨蛋白质组分析提供了机会。
