Data Sheet 1_TIE-UP-SIN: a novel method for enhanced identification of protein–protein interactions.pdf

Proteins function through complex interaction networks that govern nearly all aspects of cellular physiology. Identifying protein–protein interactions (PPIs) under native conditions remains challenging due to the transient nature of many complexes and technical limitations of conventional approaches. We present TIE-UP-SIN (Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization), a robust and reproducible method for in vivo identification of PPIs. This approach combines metabolic labeling with 15N isotopes, reversible in vivo formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN is specifically designed to preserve transient or weak interactions during purification and to quantify interaction partners using internal light/heavy peptide ratios, reducing experimental variability and increasing reproducibility across biological replicates. The method employs a triple-sample design (WT/WT, Bait/WT, Bait/Bait) to distinguish specific from non-specific interactors. Peptide-level L/H ratios are normalized against sample-specific factors, aggregated at the protein level, and statistically analyzed using moderated testing. This strategy enables reliable detection of differential PPIs across physiological states, even in organisms with limited labeling options. We demonstrate the utility of TIE-UP-SIN by mapping interaction partners of the essential housekeeping sigma factor RpoD (SigA) under control and ethanol stress conditions. Known partners such as RNA polymerase subunits (RpoA, RpoB, RpoC) were robustly enriched, while potential novel candidates, including ClpX and AcpA, were detected at lower abundance. TIE-UP-SIN offers a simple, cost-effective, and modular platform for quantitative interactome analysis and can be adapted to a wide range of bacterial and non-bacterial systems. Compared to established approaches such as label-free IP–MS or proximity-based labeling methods, TIE-UP-SIN is intended as a complementary option. Its combination of specific control, robust quantification, and suitability for low-input material provides an additional tool within the broader proteomics workflow collection.

蛋白质通过复杂的相互作用网络行使功能，该网络调控细胞生理活动的几乎所有方面。在天然条件下鉴定蛋白质-蛋白质相互作用（protein-protein interactions, PPIs）仍颇具挑战，这是因为多数复合物具有瞬态特性，且传统方法存在技术局限性。我们介绍TIE-UP-SIN（基于稳定同位素归一化的未知蛋白质靶向相互作用组实验，Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization），这是一种可在活体内鉴定蛋白质-蛋白质相互作用的稳健且可重复的方法。该方法将15N同位素代谢标记、活体内可逆甲醛交联、亲和纯化与定量质谱技术相结合。TIE-UP-SIN专为在纯化过程中保留瞬态或弱相互作用而设计，并通过内部轻/重肽段比值对相互作用伴侣进行定量，从而降低实验变异度，提升生物学重复间的可重复性。该方法采用三重样本设计（野生型/野生型、诱饵蛋白/野生型、诱饵蛋白/诱饵蛋白），以区分特异性相互作用蛋白与非特异性相互作用蛋白。肽段水平的轻/重比值会针对样本特异性因素进行归一化处理，随后在蛋白质水平进行汇总，并通过调节型检验进行统计学分析。该策略可实现在不同生理状态下可靠检测差异蛋白质-蛋白质相互作用，即便在标记手段有限的生物体中亦是如此。我们通过在对照与乙醇胁迫条件下绘制核心持家σ因子RpoD（SigA）的相互作用伴侣图谱，验证了TIE-UP-SIN的实用性。已知的相互作用伴侣如RNA聚合酶亚基（RpoA、RpoB、RpoC）得到了稳健富集，而包括ClpX与AcpA在内的潜在新型候选相互作用蛋白则以较低丰度被检测到。TIE-UP-SIN为定量相互作用组分析提供了一个简便、经济且模块化的平台，可适配多种细菌与非细菌研究系统。与无标记免疫沉淀质谱（label-free IP-MS）或基于邻近标记的方法等成熟技术相比，TIE-UP-SIN可作为一种补充性技术手段。其兼具特异性对照、精准定量以及适配低起始量样本的特性，为更广泛的蛋白质组学工作流程提供了又一实用工具。