In Vivo Chemical Cross-Linking Coupled with Reversed-Phase Co-Fractionation Mass Spectrometry Enables Protein Complex Analysis

Abstract Understanding protein–protein interactions (PPIs) and the architecture of protein complexes is essential for elucidating cellular functions and drug action mechanisms. Co-fractionation mass spectrometry (CF-MS) has emerged as an effective tool for analyzing protein complexes under near-native conditions, however, its resolution and structural fidelity are often compromised by complex dissociation during biochemical processing. Here, we present an analytical strategy that integrates in vivo formaldehyde cross-linking with reversed-phase cofractionation mass spectrometry (XL-RP-CF-MS) to identify protein complexes in living cells. Cross-linking stabilizes native interactions, preserving complex integrity during denaturing separation. Reversed-phase liquid chromatography provides high-resolution, reproducible fractionation, enabling robust detection of coelution patterns. Applying this platform to RS4;11 leukemia cells, we identified 6042 proteins and detected 1753 CORUM complexes together with 487 high-confidence EPIC-predicted complexes. The workflow also demonstrated excellent quantitative performance, exhibiting highly linear correlations between chromatographic peak areas and protein loading (R2 = 0.9972) as well as between MS signal intensities and loading amounts (R2 > 0.9). This strategy enables sensitive and physiologically relevant identification of protein complexes, offering a reliable approach for characterizing the composition of potential protein assemblies within specific biological systems.