Ultrafiltration-Enhanced Cross-Linking Mass Spectrometry for Comprehensive Analysis of Low Molecular Weight Protein Cross-Links

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.