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.