从去卵黄斑马鱼胚胎中鉴定蛋白质的实验方案。

Protocol for identification of proteins from deyolked zebrafish embryos.

作者信息

Purushothaman Kathiresan, Tneo Rwei Qing Saraphina Dianne, Ee Yong Xin, Ananthakrishnan Nithiyakala, Harshiktha Shanmugam, Vij Shubha, Lin Qingsong, Babiak Igor

机构信息

Genomics Group, Faculty of Biosciences and Aquaculture, Nord University, 8049 Bodø, Norway; School of Applied Science, Republic Polytechnic, 9 Woodlands Avenue 9, Singapore 738964, Singapore; Department of Preclinical Sciences and Pathology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway; Tropical Futures Institute, James Cook University Singapore, 149 Sims Drive, Singapore 387380, Singapore.

School of Applied Science, Republic Polytechnic, 9 Woodlands Avenue 9, Singapore 738964, Singapore.

出版信息

STAR Protoc. 2025 Jun 20;6(2):103728. doi: 10.1016/j.xpro.2025.103728. Epub 2025 Apr 5.

DOI:10.1016/j.xpro.2025.103728

PMID:40184248

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12002977/

Abstract

Zebrafish is a key model for studying vertebrate development and human diseases, but proteomic data during embryogenesis are limited due to interference from egg yolk proteins. Here, we present a protocol for the isolation, identification, and analysis of proteins from zebrafish embryos. We describe steps for dechorionation, deyolking, protein extraction, SDS-PAGE, and trypsin digestion. We then detail procedures for peptide separation using liquid chromatography-tandem mass spectrometry (LC-MS/MS), identification via ProteinPilot, and functional analysis with Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology. For complete details on the use and execution of this protocol, please refer to Purushothaman et al..

摘要

斑马鱼是研究脊椎动物发育和人类疾病的关键模型，但由于卵黄蛋白的干扰，胚胎发育过程中的蛋白质组学数据有限。在此，我们展示了一种从斑马鱼胚胎中分离、鉴定和分析蛋白质的方法。我们描述了去卵膜、去除卵黄、蛋白质提取、SDS-PAGE和胰蛋白酶消化的步骤。然后，我们详细介绍了使用液相色谱-串联质谱（LC-MS/MS）进行肽段分离、通过ProteinPilot进行鉴定以及利用京都基因与基因组百科全书（KEGG）和基因本体论进行功能分析的程序。有关本方法的使用和执行的完整详细信息，请参考Purushothaman等人的研究。