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

Area of Science:

• Proteomics
• Chemical Biology
• Molecular Cell Biology

Background:

• Protein complexes are crucial for cellular functions and drug responses.
• Traditional co-fractionation mass spectrometry (CoFrac-MS) for analyzing protein complexes is slow and labor-intensive.
• Rapid identification of drug-perturbed protein complexes is needed for drug discovery.

Purpose of the Study:

• To introduce and validate a novel, rapid chromatography-guided strategy for identifying drug-perturbed protein complexes.
• To establish the XL-CoFrac-Q-MS workflow for high-resolution separation, precise quantification, and differential analysis of protein complexes.
• To demonstrate the workflow's utility in identifying drug-target interactions and elucidating drug mechanisms.

Main Methods:

• Developed the XL-CoFrac-Q-MS workflow combining cross-linking enhanced reversed-phase liquid chromatography co-fractionation (XL-CoFrac) with ChromaQuant software for peak quantification.
• Utilized ChromaQuant for precise peak quantification (CV < 1%) and differential analysis of protein complexes.
• Applied the workflow to analyze drug responses in leukemia cells treated with a BCL-2 inhibitor (ABT-199).

Main Results:

• XL-CoFrac-Q-MS enabled rapid and precise profiling of protein complexes with high reproducibility (R > 0.98).
• The workflow successfully identified drug-perturbed protein complexes, including the BCL-2 associated complex, in response to ABT-199 treatment.
• Identified cross-linking peptides between BCL-2 and FKBP38, potentially revealing mechanisms of drug resistance.
• Pathway enrichment analysis provided insights into ABT-199-induced apoptosis.

Conclusions:

• The XL-CoFrac-Q-MS strategy offers a significant advancement for rapid identification of drug-perturbed protein complexes.
• This workflow facilitates efficient drug target screening and the elucidation of protein complex dynamics in pharmacological and pathological contexts.
• The method holds broad potential for understanding cellular responses to various perturbations.