Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Kiel 24105, Germany.
Department of Microbial Proteomics, Institute of Microbiology, University of Greifswald, Greifswald 17489, Germany.
J Proteome Res. 2021 May 7;20(5):2895-2903. doi: 10.1021/acs.jproteome.1c00115. Epub 2021 Mar 24.
The identification of proteins below approximately 70-100 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEPs), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysargiNase, and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins, with a focus on SEPs, in the archaeon . Combining the data of all proteases, we identified 63 small proteins and additional 28 SEPs with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEPs, a complete sequence coverage was achieved. Moreover, for five SEPs, incorrectly predicted translation start points or potential proteolytic processing were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach allows to improve the identification and molecular characterization of small proteins and SEPs. LC-MS data: ProteomeXchange PXD023921.
在 bottom-up 蛋白质组学中，鉴定大约 70-100 个氨基酸以下的蛋白质仍然是一项具有挑战性的任务，因为酶解产生的肽数量有限。这包括短开放阅读框编码肽 (SEP)，它们是以前未注释或替代编码的小蛋白质的子集。在这里，我们系统地研究了多种蛋白酶（胰蛋白酶、糜蛋白酶、LysC、LysargiNase 和 GluC）在 GeLC-MS/MS 分析中的使用，以提高小蛋白质的序列覆盖度和鉴定的肽数量，重点是 SEP 在古菌中。结合所有蛋白酶的数据，我们鉴定了 63 个小蛋白质和另外 28 个具有至少两个独特肽的 SEP，而仅使用胰蛋白酶可鉴定出 55 个小蛋白质和 22 个 SEP。对于 27 个小蛋白质和 12 个 SEP，实现了完整的序列覆盖。此外，对于五个 SEP，确定了错误预测的翻译起始点或潜在的蛋白水解处理，这证实了该生物之前的自上而下蛋白质组学研究的数据。结果清楚地表明，多蛋白酶方法可以提高小蛋白质和 SEP 的鉴定和分子特征。LC-MS 数据：ProteomeXchange PXD023921。
