Ana Paula Carvalho Reis, Giovanna Azevedo Celestrino, Talita Souza Siqueira, Milena De Melo Scarano Coelho, Juliana Carreiro Avila, Isabela De Oliveira Cavalcante Pimentel, Leo Kei Iwai, Pritesh Jaychand Lalwani, Vitor Manoel Silva Dos Reis, Kaique Arriel, José Ângelo Lindoso, Gil Benard, Maria Gloria Teixeira Sousa
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引用次数: 0
摘要
皮肤是人体最大的器官,也是多种疾病的发病部位。然而,考虑到皮肤组织的复杂性,关于皮肤蛋白质组学的文献数据有限。在这里,我们提出了一种适应和优化的方案,用于从人体皮肤中提取蛋白质,使用化学和机械裂解相结合的方法。为此,我们使用了含有2% SDS, 50 mM TEAB, 1%蛋白酶和磷酸酶抑制剂混合物的裂解缓冲液,以及Matrix a珠和FastPrep-24 5G均质机。为了对样品进行表征,采用SP3法(单锅,固相,样品制备)对所得蛋白质进行纯化和消化,并采用纳米液相色谱-串联质谱法进行分析。通过这种方式,我们能够从健康个体和真菌感染孢子菌病患者的皮肤样本中识别出大约6000种蛋白质。我们改进的方法可以显著丰富我们对皮肤生物学的理解,并为发现皮肤疾病的生物标志物和治疗靶点提供新的视角。
Optimized protein extraction protocol from human skin samples.
The skin is the largest organ in the body and is the site for a diverse set of diseases. Yet, given the complexity of the cutaneous tissue, there is a limited availability of data in the literature on skin proteomics. Here, we proposed an adapted and optimized protocol for the extraction of proteins from human skin, using a combination of chemical and mechanical lysis approaches. For this, we used of a lysis buffer containing 2% SDS, 50 mM TEAB, and a 1% protease and phosphatase inhibitor cocktail, in addition to Matrix A beads and a FastPrep-24 5G homogenizer. For the characterization of the samples, the obtained proteins were purified and digested using the SP3 method (Single-pot, solid phase, sample preparation), and analyzed by nano liquid chromatography coupled with tandem mass spectrometry. In this way, we were able to identify around 6000 proteins in the skin samples from healthy individuals and patients with the fungal infection sporotrichosis. Our improved methodology could significantly enrich our understanding of skin biology and provide new perspectives for the discovery of biomarkers and therapeutic targets for cutaneous diseases.