Multifaceted Proteome Analysis at Solubility, Redox, and Expression Dimensions for Target Identification.

Area of Science:

• Proteomics
• Systems Biology
• Biochemistry

Background:

• Understanding biological systems requires multifaceted proteome analysis, but mapping redox changes is challenging.
• Existing methods for proteome analysis lack the throughput and comprehensiveness to capture redox state alongside expression and solubility.

Purpose of the Study:

• To develop a high-throughput redox proteomics approach integrated with expression analysis (REX) and the Proteome Integral Solubility Alteration (PISA) assay.
• To benchmark the PISA-REX method using HCT116 cells treated with auranofin and apply it to study interferon-α (IFN-α) stimulated human monocytes and plasmacytoid dendritic cells (pDCs).

Main Methods:

• Development of the PISA-REX assay, integrating redox, expression, and solubility analyses.
• High-throughput mass spectrometry using tandem mass tag (TMTpro) for multiplexing up to four biological replicates.
• Application to cellular models including auranofin-treated HCT116 cells, IFN-α-stimulated human monocytes, and pDCs from wild-type versus Ncf1-mutant mice.

Main Results:

• PISA-REX demonstrated significant improvements over previous methods in analyzing proteome changes.
• Analysis of IFN-α-stimulated pDCs revealed that NCF1 deficiency enhances the STAT1 pathway.
• The study identified modulation of expression, solubility, and redox state of IFN-α-induced proteins in Ncf1-mutant pDCs.

Conclusions:

• The PISA-REX assay provides comprehensive, multifaceted proteome information, including redox state, expression, and solubility.
• This compact tool has the potential to become an industry standard in proteomics.
• PISA-REX opens new avenues for investigating the biology of health and disease, particularly in immune responses.