Schedlowski Maximilian, Michalik Stephan, Hoffmüller Tilly, Harms Marco, Steil Leif, Surmann Kristin, Hentschker Christian, Salazar Manuela Gesell, Völker Uwe, Reder Alexander
Department Functional Genomics, Center for Functional Genomics of Microbes, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany.
Front Microbiol. 2025 Sep 1;16:1657647. doi: 10.3389/fmicb.2025.1657647. eCollection 2025.
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 identification of PPIs. This approach combines metabolic labeling with N isotopes, reversible 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发挥功能，这些网络几乎控制着细胞生理学的所有方面。由于许多复合物的短暂性质以及传统方法的技术局限性，在天然条件下鉴定蛋白质 - 蛋白质相互作用（PPI）仍然具有挑战性。我们提出了TIE - UP - SIN（通过稳定同位素归一化对未知蛋白质进行靶向相互作用组实验），这是一种用于鉴定PPI的强大且可重复的方法。该方法将代谢性N同位素标记、可逆甲醛交联、亲和纯化和定量质谱相结合。TIE - UP - SIN专门设计用于在纯化过程中保留短暂或弱相互作用，并使用内部轻/重肽比率对相互作用伙伴进行定量，从而降低实验变异性并提高生物重复间的可重复性。该方法采用三样本设计（野生型/野生型（WT/WT）、诱饵/野生型（Bait/WT）、诱饵/诱饵（Bait/Bait））来区分特异性和非特异性相互作用体。肽水平的轻/重（L/H）比率针对样本特异性因子进行归一化，在蛋白质水平上汇总，并使用适度检验进行统计分析。这种策略能够可靠地检测不同生理状态下的差异PPI，即使在标记选项有限的生物体中也是如此。我们通过绘制在对照和乙醇应激条件下必需管家西格玛因子RpoD（SigA）的相互作用伙伴，证明了TIE - UP - SIN的实用性。已知的伙伴如RNA聚合酶亚基（RpoA、RpoB、RpoC）被大量富集，而潜在的新候选物，包括ClpX和AcpA，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单、经济高效且模块化的平台，并且可以适用于广泛的细菌和非细菌系统。与无标记免疫沉淀质谱（IP - MS）或基于邻近标记的方法等既定方法相比，TIE - UP - SIN旨在作为一种补充选项。其特异性对照、强大定量以及适用于低输入材料的组合，在更广泛的蛋白质组学工作流程中提供了一种额外的工具。
