Library · Biology & lab methods

What Protein-Protein Interaction Screening got wrong, from 12 theses

Summary written by a model from the records below: The records detail technical evaluations of biochemical, biophysical, and computational methods used to screen and characterize protein-protein interactions. Across these attempts, screening workflows failed due to non-specific binding artifacts, destabilizing sample preparation conditions, disruption of structural interfaces by truncation, and computational scoring limitations. These records come from graduate theses, 2021 to 2025, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.

12
theses
7
institutions
12
failure records
4
failure modes

Plus 5 problems the authors left open, listed at the end.

Failure modes, by theses reporting them

Select a mode to jump to its records.

Non-specific binding and surface adhesion generate high background and false signals
4
4 theses · 3 institutions · 4 records
Lysis conditions and cellular state requirements disrupt labile or transient complexes
4
4 theses · 4 institutions · 4 records
Protein truncation and fragmentation disrupt binding interfaces or overall fold
2
2 theses · 2 institutions · 2 records
Computational prediction and fractionation scoring fail to identify confident pairwise interactions
2
2 theses · 2 institutions · 2 records

Non-specific binding and surface adhesion generate high background and false signals

4 theses · 3 institutions

Assays suffered from non-specific stickiness, peptide precipitation, and plate adhesion that produced false-positive binding curves and optical artifacts. Furthermore, antibodies and peptide-based probes showed high non-specific background that obscured target enrichment.

Tried and failed

TR-FRET assay for protein-protein interactions applied to membrane-associated enzyme binding affinity. Reason: Full-length protein exhibited non-specific stickiness, yielding false-positive saturable binding curves with negative controls.

In vitro and in vivo studies of two essential MRSA cell wall synthesis enzymes · Harvard · PhD

Tried and failed

co-immunoprecipitation of endogenous proteins applied to transcription factor protein-protein interactions. Outcome: no signal. Reason: antibody non-specific binding caused high background signal present in both control and knockout lysates

B-MYB and FOXM1 drive cell division through cooperative regulation of cell cycle genes. · Harvard · PhD

Considered and rejected

Considered and rejected: Rejected peptide-based pull-down probes for proteome-wide HDAC interactome profiling due to weak, non-significant enrichment and high non-specific background compared to designer nucleosomes.

Synthesis, biochemical characterization and genetic encoding of acetyl-lysine mimicking amino acids for bromodomains and deacetylases · Tübingen · PhD

Tried and failed

fluorescence polarization competition assay for screening binding applied to macrocyclic protein-protein interaction inhibitors. Outcome: no signal. Reason: peptide precipitation and non-specific plate sticking caused elevated anisotropy and decreased total fluorescence artifacts

Generating macrocyclic inhibitors of protein-protein interactions · EPFL · PhD

Lysis conditions and cellular state requirements disrupt labile or transient complexes

4 theses · 4 institutions

Protein interactions were lost when cell lysis procedures or reducing agents disrupted weak complexes or when target proteins rapidly degraded without proteasome inhibition. Additionally, detection failed when the interaction was non-constitutive and depended on specific cellular states such as replication initiation.

Tried and failed

co-immunoprecipitation of endogenous protein complexes applied to endogenous yeast protein-protein interactions. Outcome: no signal. Reason: endogenous interaction was too weak, transient, or disrupted during cell lysis despite recombinant binding

Parsing the Dual Roles of Cu/Zn Superoxide Dismutase (Sod1) in Oxidative Stress Protection and Redox Signaling · Georgia Tech · PhD

Tried and failed

co-immunoprecipitation without proteasome inhibition applied to detecting transient protein-protein interactions. Outcome: no signal. Reason: Target protein underwent rapid proteasomal degradation in the absence of a proteasome inhibitor.

MFSD7C: A Solute Carrier Linking Heme and Calcium in Mitochondrial Energy Metabolism · MIT · PhD

Tried and failed

co-immunoprecipitation for protein-protein interaction detection applied to non-replicating cell extracts. Outcome: no signal. Reason: interaction does not occur constitutively in the absence of active replication initiation

DONSON: A novel metazoan DNA replication initiation factor · Harvard · PhD

Tried and failed

co-immunoprecipitation with reducing agent in lysis buffer applied to protein-protein interaction detection. Outcome: no signal. Reason: reducing agent in lysis buffer disrupted the protein-protein interaction complex

Modulation of innate immune signalling by salmonella effector SteE · Imperial · PhD

Protein truncation and fragmentation disrupt binding interfaces or overall fold

2 theses · 2 institutions

Terminal truncations eliminated binding signals by compromising structural integrity or removing critical contact surfaces during two-hybrid screening. Similarly, isolated domain fragments failed to interact because they lacked the binding interface required for association.

Tried and failed

Terminal truncation mapping in yeast two-hybrid applied to protein-protein interaction domain identification. Outcome: no signal. Reason: Truncations from either terminus disrupted overall structural integrity or binding interface, eliminating binding signal

Unraveling the Small Subunit Processome to tie the central pseudoknot · UT Austin · PhD

Tried and failed

co-immunoprecipitation of protein fragments applied to truncated protein-protein interaction screening. Outcome: no signal. Reason: The isolated N-terminal domain fragment lacked the binding interface required for interaction.

Conflict between bacteriophages and a mobile genetic element in bacterial immunity · MIT · PhD

Computational prediction and fractionation scoring fail to identify confident pairwise interactions

2 theses · 2 institutions

Deep learning structure prediction yielded low confidence scores and failed to locate reliable interacting residues between complex subunits. Similarly, co-fractionation mass spectrometry failed to generate high-confidence pairwise interaction scores despite observing subunit co-elution.

Tried and failed

deep learning protein-protein complex structure prediction applied to heteromeric membrane protein complexes. Outcome: no signal. Reason: Prediction yielded very low confidence scores without identifying reliable interacting residues between subunits

Characterisation of novel motility proteins in flagellar motors using cryo-electron microscopy · Imperial · PhD

Tried and failed

co-fractionation mass spectrometry for interaction scoring applied to detecting direct protein-protein interactions. Outcome: no signal. Reason: failed to yield high-confidence pairwise interaction scores despite detecting complex subunit co-elution

VerteBrain reveals novel neuronal and non-neuronal protein assemblies conserved across vertebrate evolution · UT Austin · PhD

Left open by the authors

Problems the authors named and did not get to.

Left open

Comprehensively test the structural model evaluating TPS complex stabilization via protein-protein interactions with Tsl1 or Tps3. Blocker: Requires wet-lab structural biology or biochemical assays to test protein-protein interactions in yeast

INVESTIGATING THE ROLE OF TREHALOSE METABOLISM IN SACCHAROMYCES CEREVISIAE · Cornell · PhD

Left open

Elucidate the mechanism balancing phage detection sensitivity and autoimmunity risk against host proteins in bacterial defense systems. Blocker: Requires wet-lab molecular biology experiments and protein-protein interaction assays in bacterial host cells

Mechanisms of phage detection by bacterial innate immune proteins · MIT · PhD

Left open

Investigate specific protein-protein interactions between TerC proteins (MeeF, MeeY) and secretosome components (SecDF, PrsA, FtsH). Blocker: Requires a wet lab and molecular biology assays (e.g., co-immunoprecipitation, bacterial two-hybrid) to test protein interactions.

THE FUNCTION OF TERC PROTEINS IN METAL HOMEOSTASIS · Cornell · PhD

Left open

Determine if SteE interacts with and directs phosphorylation on other host kinase substrates using kinase assays or interactome screening. Blocker: Requires wet-lab biochemical assays, protein expression, and kinase interaction screening.

Structural and Functional characterisation of SteE, a Salmonella type III secretion system effector · Imperial · PhD

Left open

Determine the binding interface, stoichiometry, cellular relevance, and kinase substrate relationship of the VRK1-PARP1 protein-protein interaction. Blocker: Requires wet-lab biochemical and cellular assays to measure physical protein interactions, stoichiometry, and phosphorylation.

Targeting VRK1: Selective Inhibitors, Chemical Probes, and Biochemical Insights · Harvard · PhD

Who has worked on this

Authors of the public theses cited on this page, most-cited first.

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