Zhang Beirong, Gong Zhou, Zhong Bowen, Liang Zhen, Zhang Yukui, Zhao Qun, Zhang Lihua
State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2025 May 13;97(18):9606-9612. doi: 10.1021/acs.analchem.5c00331. Epub 2025 Apr 30.
Low molecular weight (LMW) proteins are crucial for cellular functions, including transcription, translation, immune response, and homeostasis. However, their small size and limited lysine residues pose significant challenges in cross-linking mass spectrometry (XL-MS), resulting in low cross-linking efficiency and difficulty detecting protein interactions. To address these issues, we developed an ultrafiltration membrane-aided size exclusion chromatography (UF-SEC) strategy. By utilizing ultrafiltration membranes with progressively smaller pore sizes (ranging from 0.45 μm to 10 kDa), this method selectively removes high molecular weight proteins, enriching cross-linked LMW protein complexes and enhancing the sensitivity and specificity of XL-MS. Compared to traditional high-pH reversed-phase or strong cation exchange fractionation methods, UF-SEC provides better complementarity at the protein level with peptide fractionation methods, offering a more effective solution for identifying LMW protein complexes. Using UF-SEC, we constructed a comprehensive protein interaction network for LMW proteins (defined as <20 kDa), identifying 234 protein-protein interactions involving 77 proteins, accounting for 47.8% of the entire interaction network. This approach not only provides cross-linking distance restraints for intracellular complexes of LMW proteins but also enables scalable cross-linking evidence for PPIs, revealing potential functions such as microprotein generation from noncoding RNAs. Therefore, UF-SEC significantly enhances the capability of XL-MS to investigate LMW protein complexes, offering a powerful tool to deepen our understanding of the roles of small but crucial proteins in cellular biology.
低分子量（LMW）蛋白质对于细胞功能至关重要，包括转录、翻译、免疫反应和体内平衡。然而，它们的小尺寸和有限的赖氨酸残基在交联质谱分析（XL-MS）中带来了重大挑战，导致交联效率低且难以检测蛋白质相互作用。为了解决这些问题，我们开发了一种超滤膜辅助尺寸排阻色谱法（UF-SEC）策略。通过使用孔径逐渐减小（范围从0.45μm到10 kDa）的超滤膜，该方法选择性地去除高分子量蛋白质，富集交联的LMW蛋白质复合物，并提高XL-MS的灵敏度和特异性。与传统的高pH反相或强阳离子交换分级分离方法相比，UF-SEC在蛋白质水平上与肽分级分离方法具有更好的互补性，为鉴定LMW蛋白质复合物提供了更有效的解决方案。使用UF-SEC，我们构建了一个针对LMW蛋白质（定义为<20 kDa）的全面蛋白质相互作用网络，鉴定出涉及77种蛋白质的234种蛋白质-蛋白质相互作用，占整个相互作用网络的47.8%。这种方法不仅为LMW蛋白质的细胞内复合物提供了交联距离限制，还为蛋白质-蛋白质相互作用提供了可扩展的交联证据，揭示了潜在功能，如非编码RNA产生微蛋白。因此，UF-SEC显著增强了XL-MS研究LMW蛋白质复合物的能力，为深化我们对细胞生物学中小而关键蛋白质作用的理解提供了一个强大工具。
