Protein-Protein Interactions


Protein-Protein Interactions


Protein-Protein Interactions (PPI) provide insight into the cellular components, molecular functions, and biological processes mediated or affected by the protein of interest (POI). Research and development in the protein interaction network identifies critical proteins involved in the etiology of diseases, informs consequences to inhibition and also identifies new protein targets for drug development.


Protein protein interaction networks are the networks of protein complexes formed by biochemical events and/or electrostatic and hydrophobic forces. PPI networks serve distinct biological functions. The protein interactome describes the full repertoire of a biological system’s protein-protein-interactions (PPIs).

Rockland Immunochemicals utilizes both classic and unique approaches to elucidate PPI, including Snapshot Proteomics™, to explore cell signalingsignal transduction as well as drug design studies to understand the effects of physical contact between two or more proteins.  Our innovative approach to PPI can identify over 21,000 human targets.

 

PPI Network Analysis for Biological Context


The complexity of protein-protein interaction networks requires a systems approach using specialized analysis to comprehensively understand protein function. Critical aspects of protein function elucidated by PPI studies include:

  • Post-translational modifications (PTM) or activation deactivation events also called signed interaction networks. These include, but are not limited to phosphorylation/dephosphorylation, methylation/demethylation, acetylation/deacetylation and hydroxylation/dehydroxylation. 
  • Protein-protein interactions (PPI) including protein complex interactions and small molecule-protein interaction.
  • Drug design strategies including epi-proteomic phenotypic screens, comprehensive mechanism of action (MOA) and safety/efficacy profiling using control and drug treated or perturbagen treated cell lysates.
  • Enzyme action studies including kinase based assays and enzyme-substrate pairing studies. 
  • Antibody specificity studies including autoimmune disease and autoantibody studies. 
  • Biomarker discovery in clinical samples for molecular diagnostics (MDx), companion diagnostics (CDx), theranostics (THx) or cohort stratification including response to therapy.

 


 Basic Science   Drug Discovery  Biomarker Discovery Drug Repurpusing

Our Approach to PPI Analysis


Traditional proteomics breaks the proteins present in a sample, biochemically separates the pieces, and identifies only the most abundant components to provide information on gene expression. This type of analysis is only capable of identifying about 8,000 human targets with an unacceptably low rate of data reproducibility estimated at 60%. Complexes and networks are destroyed and most biological information about interactions and activities is lost in the process. The “top down” proteomics approach reveals no information regarding the activation state of the target, despite utilizing tandem mass spectrometry.

Fortunately our innovative approach to PPI called Snapshot Proteomics™ preserves the majority of biological information resulting in significant discoveries about the activity and interaction of proteins present in samples. Furthermore, our technology can identify over 21,000 human targets and demonstrates a high rate of data reproducibility estimated at >95%. This technology provides the deepest, most reliable, and most biologically relevant information about a sample you could possibly gather. It provides comprehensive proteomic information about protein-protein interactions, enzyme-substrate identifications, and phenotypic interventions like drug treatment.

 

 


 

Snapshot Proteomics™


Snapshot Proteomics™ uses microarrays containing more than 20,000 purified human proteins. Treating the microarray with a biological sample, such as a cell lysate, results in changes to relevant protein targets on the array including interactions with proteins and enzymes. This occurs because both the array and test samples are maintained in a near native state, allowing enzymes and proteins to remain properly folded, modified, in-complex, and functionally active. The presence of competing substrates and antagonistic activities within the sample adds physiological relevance to the events observed on the array.

The beginning of Snapshot Proteomics™ is analogous to a massively parallel co-IP in which specific proteins are introduced and biochemically assayed within lysates (or, also in this case, clinical samples). The readout is analogous to a massively parallel Western blot in that spatially separated proteins at unique locations on the arrays can be identified to interact with sample components and individual protein changes can be reported at the molecular level.

Inquire about Snapshot Proteomics™ here:
 
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Services Offered

 

Snapshot Proteomics™ is a core technology that powers several services, each tailored to diverse scientific needs. You will receive:

  • Primary data.
  • Proprietary bioinformatics analyses to reveal the underlying biology.
  • A full report with detailed methods and explanation of results.
  • Consultation with our scientists who will personally present their findings, and provide technical support should additional questions arise.

Use Snapshot Proteomics™ to better inform your science, basic or applied.

 

 
 Snapshot-Figure-1 

 

Figure 1. Enzyme-substrate identification. Left image from array treated with cells knocked-out for a specific E3 ligase. Image on right from an identical array treated with control cells containing WT levels of that E3. Red box indicates a novel substrate of the ligase.


Materials and Methods

 

Provided by client:  cells, lysates, proteins of interest, drugs of interest, perturbagens, or other relevant materials required to complete the study according to mutual agreement.  In some instances, these materials can be produced or provided by Rockland Immunochemicals Inc. under separate agreement.

 

After completing the experiments and analysis Rockland Immunochemicals Inc. will provide the following:

 

  • raw images of the arrays
  • quantitation of all proteins present on all arrays, e.g. for two arrays 21,000 proteins printed in duplicate would yield 84,000 total data points
  • twice-normalized data (for biological controls and Loess normalized for technical sources of error)
  • a comprehensive list of significant effects or interactions
  • a list of high confidence effects or interactions corrected for false discovery (FDR)
  • a complete report detailing our methods and bioinformatics analysis of data

Upon completion we will schedule a call to review and discuss our findings with your staff.
 

 

The Snapshot Proteomics™ Approach to PPI can aid Basic Science by:

  • Determining what proteins interact with the protein of interest (POI).
  • Identifying what substrates are acted on by an enzyme of interest.
  • Revealing what enzymes regulate the POI.
  • Determining the precise specificity of an antibody including both on target and off target effects.
  • Examining how the proteome responds to overexpression/deletion/mutation of POI.


The Snapshot Proteomics™ Approach to PPIN can aid Drug Discovery by:

  • Identifying exactly what proteins are affected by a drug compound or therapeutic antibody.
  • Determine how the cellular proteome responds to the presence of a drug or perturbation.
  • Elucidate the MOA of a drug that may drive the intended effect and side-effects.

 Snapshot-Figure-2 

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Figure 2: Sorafenib-treated cells compared to DMSO-treated control cells. A) Phosphorylation of PDGFR and several MAPK members (red arrows) was decreased as expected: YAP1 and its effector kinases were increased in phosphorylation, consistent with being the driver of apoptosis as previously suggested. B) Changes to phosphorylation across the proteome in response to Sorafenib were examined for the presence of ontologies; those found were highly consistent with expectations for an anti-cancer drug operating through membrane receptors (note the presence of ubiquitin pathway, another PTM pathway known to drive cancer).

 

Snapshot Proteomics™ for Biomarker Discovery


 For drug development, truly useful biomarkers are specific to the compound, not the intended disease or molecular target. Particularly in cases where traditional techniques (Genome-Wide Association Studies, Next-Gen Sequencing, and Tandem Mass Spectrometry) have failed, Snapshot Proteomics™ can deliver novel and useful markers custom tailored to your project. 

CASE STUDY: Utilized by leading pharmaceutical company, the Snapshot Proteomics™ approach to protein-protein interaction networks was used to successfully discover novel pharmacodynamic (PD) biomarkers in human sera, for an investigational analgesic known to inhibit prostaglandin synthesis. The compound was in clinical trial, and previous attempts with alternative technologies were unsuccessful. 

 Snapshot-Figure-3 

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Figure 3.  Snapshot Proteomics™ used to identify PD biomarkers. Four different buffer conditions were evaluated, ranging from most natural (B1) to most chemically manipulated (B4). Human sera were used to identify proteomic changes in ubiquitylation (UB) and phosphorylation (PO4) in response to drug. Three biomarkers were identified in common to all samples tested; all markers were consistent with the drug’s intended use and known MOA. Markers identified were: phosphorylated UCN3, a protein known to bind G-protein receptors and linked to pain; phosphorylated GAGE1, a serum antigen involved in response to foreign agent and ubiquitylated SEPT6, a protein found in synaptic vesicles and the axon terminus. FDR stands for false discovery rate.

 


Snapshot Proteomics™ for Drug Repurposing

 

Because most proteins engage multiple molecular interactions throughout their lifecycle, even a perfectly precise drug will have unintended consequence to the cell. Like ripples through a pond, these other proteins likewise engage different molecular interactions that will, in turn, be affected by alteration to an upstream event. Whether the drug targets housekeepers, enzymes, receptors, or transcription factors, the full scope of effects is best viewed from an epi-proteomic perspective. That is because these PTM-driven events precede and drive all other cellular responses including transcription, translation and degradation (which themselves can be viewed with more crude levels of analysis that miss the full scope of activity). 

 
Of course, most drugs are not perfectly precise in their targets, making such analysis even more informative. By identifying the off-targets, and more importantly the consequences of hitting them, our proprietary proteo-genomic analysis of the epi-proteomic changes in response to drug enables a preclinical view into the cellular effects most affected by a drug. Such comprehensive MOA can identify opportunities for reuse of approved drugs, or to speed orphan drug approval. Not all ripples in the pond are negative: and unless a drug has been screened against all possible targets or been administered to patients suffering all possibilities of diagnosed co-morbidity in which only one condition was being treated, the effects remain to be discovered. But only if looked for properly.



Related Protein-Protein Interaction Services   Catalog #
Protein-Protein Interaction Studies utilizing Cell Lysates (SPI-ID)    PPI-001-CL
     
Protein-Protein Interaction Studies utilizing Recombinant Protein (PPI-ID)   PPI-001-RP
     
Substrate Identification Studies utilizing Cell Lysates (SS-ID)   PPI-002-CL
     
Substrate Identification Studies utilizing Recombinant Protein (Sub-ID)   PPI-002-RP
     
Drug Profiling utilizing cell lysates, serum, plasma or other analytes (PSE-1). Single channel detection of one PTM   PPI-003-01
     
Drug Profiling utilizing cell lysates, serum, plasma or other analytes (PSE-2). Dual channel detection of two PTMs   PPI-003-02
     
Biomarker Discovery utilizing Clinical Samples (BMD)   PPI-004
     
Antibody Specificity Profiling of Purified Antibody (ASP)    PPI-005
     
Autoantibody Profiling for Discovery Related to Autoimmune Disease (AASP)   PPI-006


PPI Services



Related Products and Services:

Custom Antibody Production   Mass Spectrometry
Cell Culture Services   Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
Purification Services   Next Generation Sequencing (NGS or Next Gen Seq)
Secondary Antibodies   Surface Plasmon Resonance (SPR)
Blocking Buffers   In Vitro Diagnostic Services and Solutions
Enzyme Substrates    

 For all assay development services and information requests please inquire with one of our experts.

 

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