Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
J Proteomics. 2019 Mar 1;194:89-98. doi: 10.1016/j.jprot.2018.12.011. Epub 2018 Dec 13.
Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with advantages such as reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or to single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions. This simple and straightforward strategy facilitated complete incorporation of amino acids into the bacterial proteome with good accuracy. High labeling efficiency can be achieved in different bacteria by slightly modifying the supplementation of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics. SIGNIFICANCE: Amino acid conversion in bacteria decreases labeling efficiency, limiting the application of Stable isotope labeling with amino acids in cell culture (SILAC) in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that high concentrations of isotope-labeled (heavy) and natural (light) amino acids facilitate full incorporation of amino acids into the bacterial proteome with good reproducibility. This improved double labeling SILAC technique using medium supplemented with high concentrations of amino acids is suitable for quantitative proteomics research on both gram-positive and -negative bacteria, facilitating the broad application of quantitative proteomics in bacterial studies.
稳定同位素标记相对和绝对定量技术（SILAC）是一种强大的蛋白质组学方法，具有可重复性和易于处理等优点。该方法常用于分析哺乳动物细胞。然而，细菌中的氨基酸转换会降低标记效率和定量准确性，限制了 SILAC 在细菌蛋白质组学中的应用，只能用于营养缺陷型细菌或赖氨酸的单一标记。在本研究中，我们发现，在 SILAC 基本培养基中添加高浓度的同位素标记（重）和天然（轻）氨基酸可以有效地抑制复杂的氨基酸转换。这种简单直接的策略可以促进氨基酸完全掺入细菌蛋白质组，具有良好的准确性。通过稍微修改培养基中氨基酸的补充，可以在不同的细菌中实现高标记效率，从而促进 SILAC 技术在细菌蛋白质组学中的广泛应用。意义：细菌中的氨基酸转换会降低标记效率，限制稳定同位素标记相对和绝对定量技术（SILAC）在细菌蛋白质组学中的应用只能用于营养缺陷型细菌或赖氨酸的单一标记。在本研究中，我们发现高浓度的同位素标记（重）和天然（轻）氨基酸有助于氨基酸完全掺入细菌蛋白质组，具有良好的重现性。这种使用高浓度氨基酸补充培养基的改进的双重 SILAC 技术适用于革兰氏阳性和阴性细菌的定量蛋白质组学研究，促进了定量蛋白质组学在细菌研究中的广泛应用。
