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首页> 外文期刊>Journal of Molecular Biology Research >Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry
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Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

机译：蛋白质组学方法从血链球菌中提取细胞质蛋白的质谱分析

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Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis.
机译：血链球菌是常见的口腔早期定居者，也是传染性心内膜炎的机会病原体。提取该细菌的可溶性蛋白质组可以提供有关不同生长和胁迫条件下生理动态变化的深刻见解，从而将“蛋白质组学特征”定义为治疗干预的目标。在此协议中，我们描述了一种经过实验验证的方法，可从血链球菌SK36菌株中提取最大的胞质蛋白。采用了优化的缓冲液和超声处理步骤，可以打破厚壁细胞壁屏障并最大程度地减少细胞内蛋白质组变性的方法组合。使用Pierce BCA蛋白定量测定法对提取的蛋白质组进行定量，并通过考马斯亮蓝染色对蛋白条带进行宏观评估。最后，通过Synapt G2Si质谱仪对提取的蛋白质进行高分辨率检测，然后通过Progenesis QI进行无标记的相对定量。总之，用于蛋白质组学提取和分析可溶性蛋白质的管道为解密血链球菌的生物学复杂性提供了基本工具。

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