Karunadasa Sumudu S, Grismer TaraBryn S, Reyes Andres V, Zhai Wenxuan, Xu Shou-Ling
Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA.
Carnegie Mass Spectrometry Facility, Carnegie Institution for Science, Stanford, CA, USA.
Methods Mol Biol. 2025;2953:127-141. doi: 10.1007/978-1-0716-4694-6_9.
Proximity labeling mass spectrometry (PL-MS) is a powerful technique for mapping protein-protein interactions (PPIs), subcellular and cell type-specific proteomes, and protein-RNA and protein-DNA interactions. Direct detection of biotinylated peptides is critical for the accurate characterization of proximity-tagged proteins. A peptide-level enrichment workflow is advantageous as it facilitates the efficient removal of free biotin prior to enrichment, thus allowing for the use of higher concentrations of biotin-an important advantage in certain plant studies. This chapter provides a detailed, step-by-step protocol for sample preparation, including enrichment methods using both anti-biotin antibody-based and streptavidin-based affinity beads. Additionally, it offers an overview of software tools crucial for the reliable identification and quantification of biotinylated peptides.
邻近标记质谱法（PL-MS）是一种用于绘制蛋白质-蛋白质相互作用（PPI）、亚细胞和细胞类型特异性蛋白质组以及蛋白质-RNA和蛋白质-DNA相互作用图谱的强大技术。直接检测生物素化肽对于准确表征邻近标记的蛋白质至关重要。肽水平的富集工作流程具有优势，因为它有助于在富集之前有效去除游离生物素，从而允许使用更高浓度的生物素——这在某些植物研究中是一个重要优势。本章提供了详细的、逐步的样品制备方案，包括使用基于抗生物素抗体和基于链霉亲和素的亲和珠的富集方法。此外，它还概述了对于可靠鉴定和定量生物素化肽至关重要的软件工具。
