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Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a...
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The function of proteins depends on their native three-dimensional structure, which is dictated by the amino acid sequence of the specific protein. Folding of the polypeptide chain takes place under specific conditions that energetically favor the folded conformation. In contrast, protein denaturation occurs spontaneously under unfavorable conditions that disrupt the integrity of the folded conformation. Thus, the chemical and physical environment of a protein, such as significant changes in pH...
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Exploring How Workflow Variations in Denaturation-Based Assays Impact Global Protein-Protein Interaction Predictions.

Tavis J Reed1, Laura M Haubold2, Josiah E Hutton2

  • 1Lewis-Sigler Institute for Integrative Genomics, Princeton University, Carl Icahn Laboratory, Princeton, New Jersey, USA; Department of Computer Science, Princeton University, Princeton, New Jersey, USA; Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

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Summary
This summary is machine-generated.

Denaturation-based assays like TPCA and I-PISA reveal distinct protein-protein interaction (PPI) networks. Incorporating insoluble fractions and optimizing workflows significantly enhances PPI landscape mapping and reduces sample needs.

Keywords:
interaction networksion-based proteome-integrated solubility alterationprotein–protein interactionsproteomicsthermal proximity coaggregationtimsTOF Ultra

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Area of Science:

  • Proteomics
  • Systems Biology
  • Biochemistry

Background:

  • Protein-protein interactions (PPIs) are crucial for cellular functions.
  • Denaturation-based assays like thermal proximity coaggregation (TPCA) and ion-based proteome-integrated solubility alteration (I-PISA) are used to map global PPI networks.
  • Understanding how different denaturation methods influence PPI network characterization is essential.

Purpose of the Study:

  • To experimentally and computationally characterize the effect of denaturation-based assays on PPI network mapping.
  • To compare PPI networks identified by TPCA and I-PISA, including overlapping and distinct protein sets.
  • To assess the impact of protein properties and subcellular localization on PPI detection within these workflows.

Main Methods:

  • Generation of paired TPCA and I-PISA datasets.
  • Experimental and computational analysis of soluble and insoluble protein fractions.
  • Assessment of protein physical properties (size, complexity, hydrophobicity) and subcellular localization.
  • Evaluation of sample amount reduction and alternative quantification methods (label-free DIA vs. TMT DDA).

Main Results:

  • Distinct PPI networks are captured by TPCA and I-PISA, influenced by protein properties and localization.
  • Insoluble fractions significantly expand the detectable PPI landscape.
  • Integrating data from soluble and insoluble fractions enhances network informativeness.
  • TPCA performance remains robust with a 500x reduction in sample input.
  • Label-free DIA TPCA offers performance comparable to TMT DDA TPCA.

Conclusions:

  • Denaturation-based assays provide complementary insights into PPI networks.
  • The inclusion of insoluble fractions is critical for comprehensive PPI mapping.
  • Workflow optimization, including sample reduction and label-free quantification, offers practical improvements for global PPI network analysis.