Need for speed: Advances in the era of high throughput interaction proteomics

Protein-protein interactions are central to virtually all biological processes, forming intricate networks that operate in a highly regulated manner. These interactions are not permanent but rather continuously adapt to environmental changes, developmental cues, or disease-related stress. Understanding which protein interactions are present in a specific cellular state and how they adapt to specific stimuli is one of the long-standing goals of modern systems biology. Mass spectrometry-based proteomics has emerged as the primary tool for charting these networks. Over the past two decades, continuous advances in instrumentation, sample preparation, and data analysis have enabled researchers to explore the protein interaction landscape with increasing depth and accuracy. This has led to important discoveries in areas ranging from fundamental cell signaling to the identification of new therapeutic targets. We present the current state of MS-based protein interaction analysis, focusing on the three most widely utilized approaches: affinity purification, proximity labeling and co-fractionation mass spectrometry. For each we discuss the fundamental approach, technical considerations, limitations and highlight the potential integration with future technologies and datasets. Recent innovations such as short-gradient chromatography and faster data acquisition have further improved sensitivity and throughput. Together, these developments are bringing researchers closer to mapping the dynamic, context-dependent architecture of protein networks in unprecedented detail.