对细胞和组织蛋白质组学的样品制备方案进行可靠评估。

IF 2.5 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochimica et biophysica acta. Proteins and proteomics Pub Date : 2024-06-27 DOI:10.1016/j.bbapap.2024.141030
Francielle Aguiar Gomes, Douglas Ricardo Souza Junior, Mariana Pereira Massafera, Graziella Eliza Ronsein
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引用次数: 0

摘要

在蛋白质组学研究中,结果的可靠性和可重复性取决于良好执行的蛋白质提取和消化方案。在此,我们系统地比较了三种成熟的巨噬细胞消化方法,即过滤辅助样品制备(FASP)、溶液中消化和凝胶中消化方案。我们还比较了冻干和人工裂解提取肝组织蛋白的方法,每种方法都使用脱氧胆酸钠(SDC)或基于 RIPA 的裂解缓冲液进行测试。就巨噬细胞系而言,使用钝化过滤单元的 FASP 在鉴定肽和蛋白质的数量方面优于其他测试方法。不过,仔细的标准化工作表明,所有三种方法都能在广泛的起始材料范围内(甚至从 1 μg 蛋白质开始)得出可靠的结果。重要的是,所有样品制备方案都确定了日间和日内差异。因此,溶液中、凝胶中和 FASP 方案的日间 CV 中值分别为 10%、8% 和 9%,与日内分析的 CV 中值(分别为 9%、8% 和 8%)非常相似。此外,FASP 消化法有 80% 的蛋白质的 CV 值低于 25%,紧随其后的是凝胶消化法(78%)和溶液样品制备法(72%)。在组织蛋白质组学方面,人工裂解和冻干两种方法的蛋白质组覆盖率和重现性相似,但蛋白质提取的效率取决于所用的裂解缓冲液,RIPA 缓冲液的效果更好。总之,尽管每种样品制备方法都有其自身的特点,但只要认真规范样品制备工作流程,它们都能成功地进行蛋白质组学实验。
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Robust assessment of sample preparation protocols for proteomics of cells and tissues

In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 μg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.

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来源期刊
CiteScore
8.00
自引率
0.00%
发文量
55
审稿时长
33 days
期刊介绍: BBA Proteins and Proteomics covers protein structure conformation and dynamics; protein folding; protein-ligand interactions; enzyme mechanisms, models and kinetics; protein physical properties and spectroscopy; and proteomics and bioinformatics analyses of protein structure, protein function, or protein regulation.
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