Hubei Key Lab of Genetic Regulation & Integrative Biology, School of Life Sciences, Central China Normal University, No. 152 Luoyu Road, Wuhan 430079, PR China.
Hubei Key Lab of Genetic Regulation & Integrative Biology, School of Life Sciences, Central China Normal University, No. 152 Luoyu Road, Wuhan 430079, PR China.
J Proteomics. 2021 Jan 6;230:103965. doi: 10.1016/j.jprot.2020.103965. Epub 2020 Sep 3.
The small proteins and short open reading frames encoded peptides (SEPs) are of fundamental importance because of their essential roles in biological processes. However, the annotation or identification of them is challenging, in part owing to the limitation of the traditional genome annotation pipeline and their inherent characteristics of low abundance and low molecular weight. To discover and characterize SEPs in Hep3B cell line, we developed an optimized peptidomic assay by combining different peptide extraction and separation methods. The organic solvent precipitation method in peptidomic showed promotion in the enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high-quality MS/MS spectra. Furthermore, different strategies exhibited good complementarity in improving the total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Results in this work provide robust evidence to date that the human proteome is more complicated than previously appreciated, and this will be a benefit to discoveries of proteins without function annotation. SIGNIFICANCE: In this work, methods were optimized to identify SEPs in Hep3B. The organic solvent precipitation presents promotion in enrichment of low molecular proteins or peptides, and the data clearly showed a beneficial effect from the reduction of sample complexity, resulting in high quality MS/MS spectra. Different strategies exhibited good complementarity in improving total amount of small proteins and their sequence coverage. In total, 1192 proteins within less than 100 amino acids were identified, including 271 newly discovered SEPs that been annotated in the OpenProt database and 147 SEPs of them encoded from ncRNA or lincRNA. Furthermore, 22 SEPs generated from the uORF may has potential effect in translation control, and 149 newly identified SEPs have known functional domains or cross-species conservation. Results in this work present robust evidence for the coding potential of the ignored region of human genomes and may provide additional insights into tumor biology.
小分子蛋白和短开放阅读框编码肽（SEPs）具有重要的基础作用，因为它们在生物过程中具有重要作用。然而，它们的注释或鉴定具有挑战性，部分原因是传统的基因组注释管道的限制及其低丰度和低分子量的固有特性。为了在 Hep3B 细胞系中发现和表征 SEPs，我们通过结合不同的肽提取和分离方法开发了一种优化的肽组学分析方法。肽组学中的有机溶剂沉淀法在低分子量蛋白质或肽的富集中表现出促进作用，数据清楚地显示出减少样品复杂性的有益效果，从而产生高质量的 MS/MS 谱。此外，不同的策略在提高小蛋白的总量及其序列覆盖率方面表现出良好的互补性。总共鉴定了小于 100 个氨基酸的 1192 种蛋白质，包括在 OpenProt 数据库中注释的 271 种新发现的 SEPs 和 147 种来自 ncRNA 或 lincRNA 的 SEPs。这项工作的结果提供了迄今为止强有力的证据，证明人类蛋白质组比以前认为的更复杂，这将有助于发现没有功能注释的蛋白质。
在这项工作中，方法被优化以鉴定 Hep3B 中的 SEPs。有机溶剂沉淀法在低分子量蛋白质或肽的富集中表现出促进作用，数据清楚地显示出减少样品复杂性的有益效果，从而产生高质量的 MS/MS 谱。不同的策略在提高小蛋白的总量及其序列覆盖率方面表现出良好的互补性。总共鉴定了小于 100 个氨基酸的 1192 种蛋白质，包括在 OpenProt 数据库中注释的 271 种新发现的 SEPs 和 147 种来自 ncRNA 或 lincRNA 的 SEPs。此外，来自 uORF 的 22 个 SEPs 可能在翻译控制中具有潜在的影响，而 149 个新鉴定的 SEPs具有已知的功能域或跨物种保守性。这项工作的结果为人类基因组中被忽视区域的编码潜力提供了强有力的证据，并可能为肿瘤生物学提供更多的见解。
