Tola Adesola Julius, Missihoun Tagnon D
Groupe de Recherche en Biologie Végétale (GRBV), Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, 3351 boul. des Forges, Trois-Rivières, QC, G9A 5H7, Canada.
Planta. 2023 Feb 19;257(3):62. doi: 10.1007/s00425-023-04083-6.
Ammonium sulfate is well known to salt out proteins at high concentrations. The study revealed that it can serve to increase by 60% the total number of identified carbonylated proteins by LC-MS/MS. Protein carbonylation is a significant post-translational modification associated with reactive oxygen species signaling in animal and plant cells. However, the detection of carbonylated proteins involved in signaling is still challenging, as they only represent a small subset of the proteome in the absence of stress. In this study, we investigated the hypothesis that a prefractionation step with ammonium sulphate will improve the detection of the carbonylated proteins in a plant extract. For this, we extracted total protein from the Arabidopsis thaliana leaves and subjected the extract to stepwise precipitation with ammonium sulfate to 40%, 60%, and 80% saturation. The protein fractions were then analyzed by liquid chromatography-tandem mass spectrometry for protein identification. We found that all the proteins identified in the non-fractionated samples were also found in the prefractionated samples, indicating no loss was incurred during the prefractionation. About 45% more proteins were identified in the fractionated samples compared to the non-fractionated total crude extract. When the prefractionation steps were combined with the enrichment of carbonylated proteins labeled with a fluorescent hydrazide probe, several carbonylated proteins, which were unseen in the non-fractionated samples, became visible in the prefractionated samples. Consistently, the prefractionation method allowed to identify 63% more carbonylated proteins by mass spectrometry compared to the number of carbonylated proteins identified from the total crude extract without prefractionation. These results indicated that the ammonium sulfate-based proteome prefractionation can be used to improve proteome coverage and identification of carbonylated proteins from a complex proteome sample.
众所周知，硫酸铵在高浓度时能使蛋白质盐析。该研究表明，它可使通过液相色谱-串联质谱法（LC-MS/MS）鉴定出的羰基化蛋白质总数增加60%。蛋白质羰基化是一种重要的翻译后修饰，与动植物细胞中的活性氧信号传导相关。然而，检测参与信号传导的羰基化蛋白质仍然具有挑战性，因为在没有应激的情况下，它们仅占蛋白质组的一小部分。在本研究中，我们探讨了一个假设，即采用硫酸铵进行预分级步骤将改善植物提取物中羰基化蛋白质的检测。为此，我们从拟南芥叶片中提取了总蛋白，并将提取物用硫酸铵逐步沉淀至饱和度为40%、60%和80%。然后通过液相色谱-串联质谱法分析蛋白质级分以进行蛋白质鉴定。我们发现，在未分级样品中鉴定出的所有蛋白质在预分级样品中也都能找到，这表明在预分级过程中没有造成损失。与未分级的总粗提物相比，分级样品中鉴定出的蛋白质多约45%。当预分级步骤与用荧光酰肼探针标记的羰基化蛋白质富集相结合时，一些在未分级样品中未见到的羰基化蛋白质在预分级样品中变得可见。一致地，与未进行预分级从总粗提物中鉴定出的羰基化蛋白质数量相比，预分级方法通过质谱法能多鉴定出63%的羰基化蛋白质。这些结果表明，基于硫酸铵的蛋白质组预分级可用于提高蛋白质组覆盖率，并从复杂的蛋白质组样品中鉴定羰基化蛋白质。
