A Chromatography-Guided Co-Fractionation Mass Spectrometry Strategy for Rapid Profiling of Drug-Perturbed Protein Complexes

Protein complexes are central to cellular function and respond rapidly to pharmacological perturbations. Co-fractionation mass spectrometry (CoFrac-MS) is widely employed to analyze protein complexes by analyzing individual chromatographic fractions, but it is labor-intensive and slow. To address these challenges, we introduce a chromatography-guided strategy enabling rapid identification of drug-perturbed protein complexes. It combines cross-linking enhanced reversed phase liquid chromatography cofractionation (XL-CoFrac) for high-resolution separation with ChromaQuant, a custom tool for precise peak quantification and differential analysis (https://hplcfdu.shinyapps.io/ChromaQuant/). Subsequent targeted MS analyses, guided by ChromaQuant, collectively establish the XL-CoFrac-Q-MS workflow. In proof-of-concept studies, we first adopted XL-CoFrac to MCF7 cells and profiled representative protein complexes. ChromaQuant demonstrated exceptional precision, achieving coefficients of variation below 1% and replicate correlations exceeding 0.98. Furthermore, we analyzed RS4;11 leukemia cells treated with increasing concentrations of the BCL-2 inhibitor ABT-199 using the XL-CoFrac-Q-MS workflow. Seven chromatographic peaks that changed consistently with the drug concentration were selected to be identified by this approach. MS analysis of these peaks revealed cross-linked peptides from the BCL-2 associated protein complex. Specially, cross-linking peptides between BCL-2 and FKBP38 may shed light on the mechanisms underlying resistance to ABT-199. Further pathway enrichment analysis provides new insights into the molecular mechanisms driving ABT-199 induced apoptosis. Collectively, the XL-CoFrac-Q-MS strategy holds significant potential for broad applications, including rapid screening of drug targets and elucidation of protein complex dynamics across various pharmacological and pathological conditions.