Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, P.R. China.
Department of Chemistry, University of California, Riverside, California 92521, United States.
Anal Chem. 2021 Apr 27;93(16):6403-6413. doi: 10.1021/acs.analchem.0c05393. Epub 2021 Apr 15.
Nascent proteome is crucial in directly revealing how the expression of a gene is regulated on a translation level. In the nascent protein identification, puromycin capture is one of the pivotal methods, but it is still facing the challenge in the deep profiling of nascent proteomes due to the low abundance of most nascent proteins. Here, we describe the synthesis of puromycin-modified silica microspheres (PMSs) as the sorbent of dispersive solid-phase microextraction and the establishment of the PMS-based nascent proteomics (PMSNP) method for efficient capture and analysis of nascent proteins. The modification efficiency of puromycin groups on silica microspheres reached 91.8% through the click reaction. After the optimization and simplification of PMSNP, more than 3500 and 3900 nascent proteins were rapidly identified in HeLa cells and mouse brains within 13.5 h, respectively. The PMSNP method was successfully applied to explore changes in the translation process in a biological stress model, namely, the lipopolysaccharide-stimulated HeLa cells. Biological functional analyses revealed the unique characters of the nascent proteomes and exhibited the superiority of the PMSNP in the identification of low abundance and secreted nascent proteins, thus demonstrating the sensitivity and immediacy of the PMSNP method.
新生蛋白质组在直接揭示基因在翻译水平上的表达调控方式方面至关重要。在新生蛋白质鉴定中，嘌呤霉素捕获是一种关键方法，但由于大多数新生蛋白质的丰度较低，它在新生蛋白质组的深度分析中仍然面临挑战。在这里，我们描述了合成了经过修饰的嘌呤霉素的二氧化硅微球（PMSs）作为分散固相微萃取的吸附剂，并建立了基于 PMS 的新生蛋白质组学（PMSNP）方法，用于高效捕获和分析新生蛋白质。通过点击反应，嘌呤霉素基团在二氧化硅微球上的修饰效率达到了 91.8%。经过 PMSNP 的优化和简化，在 13.5 小时内，分别在 HeLa 细胞和小鼠脑中快速鉴定出了超过 3500 和 3900 种新生蛋白质。该 PMSNP 方法成功应用于探索生物应激模型中翻译过程的变化，即脂多糖刺激的 HeLa 细胞。生物学功能分析揭示了新生蛋白质组的独特特征，并展示了 PMSNP 在鉴定低丰度和分泌性新生蛋白质方面的优越性，从而证明了 PMSNP 方法的灵敏度和即时性。
