Proteomics Contract Research

• Protein biomarker discovery
• Protein Identification
• Protein Capture & validation
• Protein interaction detection
• Protein affinity measurements

Overview

Biomarkers provide powerful tools in diagnosis of disease, novel target identification, and can impact all levels of the drug development process. Our contract research offering provides our clients with the means to identify novel differentially accumulated protein biomarkers from biological tissues or fluids using state of the art technology, including MALDItof/SELDItof and ESItof mass spectrometry and surface plasmon resonance.

• We screen our client’s samples with a number of established and/or proprietary standard operating protocols in order to identify candidate biomarkers.
• The candidate biomarkers are then validated using selected procedures (chosen on the basis of their optimal detection sensitivity) and applied to a larger population of samples for statistical analysis.
• Once a biomarker has been validated, we will identify the protein from sequence databanks using protein fingerprinting techniques. After identification, it is often judicious to confirm the finding by a direct assay, such as an antibody-antigen or alternative protein-ligand assay.
• We offer the development of novel protein capture assays to remove any residual doubt over the authenticity of the biomarker identification. Using the purified biomarker as bait, we will perform protein interaction analysis, mapping and identifying protein-protein interactions. As these proteins are associated with the biomarker, they too may shed crucial light on the physiological basis of the association between the biomarker and the pathogenesis in question.
• Using surface plasmon resonance, we can monitor protein association kinetics for the identified biomarker. Using multiplex SPR imaging arrays, we can provide protein arrays for multiple, parallel analysis of biomarker detection and quantification.

We offer our standard and proprietary techniques on a fee-for-service basis, where our clients retain full ownership of the data produced by the study and full intellectual property rights on the identity of any biomarker discovered therein.

Protein Biomarker Discovery                                         

Standard procedures

STEP 1: Pre-fractionation

• Optional: Serum can be de-albuminated using Cibacron Blue chromatography or using anti-albumin antibodies (human only).
• IEX fractionation: samples from complex mixtures are fractionated using strong anion ion exchange support into 4 - 6 sub-fractions.
• Other novel pre-fraction procedures are possible from a wide range of chromatographic media, including proprietary biomarker capture & display matrices (click here for more details).

 STEP 2: MS Analysis

• Fractionated protein samples are subjected to SELDItof MS analysis using Ciphergen ProteinChip arrays.
• Alternative MS analysis by direct MALDI-tof  (useful for large scale early biomarker probing).
• Several surface chemistries are available for analysis including hydrophobic, weak or strong anion/cation exchange, normal phase, IMAC (immobilised metal affinity chromatography) with Cu^2+, Ni²⁺, etc (or Ga³⁺ for phosphopeptides).
• MS analysis is performed with two matrices (CHCA or SPA) to cover the peptide to large protein range (500 Da to 200 kDa).

STEP 3: Data mining

• Multiple sample profiles are analysed for similarity and differences in their expression levels, identifying differences in the protein peaks and sorting them as a function of multiple sample groups. 

STEP 4: Data Analysis

• Optimal conditions are chosen from the above based on biomarker detection sensitivity and the number of candidate biomarkers found. A larger population of samples is screened and the data submitted to statistical analysis.

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Protein Identification

• Biomarkers are concentrated and partially purified by conventional liquid chromatography coupled to MS (LC-MS).
• Samples then are separated on 1D SDS PAGE gels and the gel slice containing the protein of interest is excised and digested with sequencing grade trypsin to provide a protein fingerprint consisting of defined peptide masses with C-terminal lysine or arginine residues.
• Optional: Chemical modification of lysine may be performed to enhance the detection of peptides terminating in these residues.
• The peptides m/z values are detected by MS and submitted for analysis to the protein sequence databanks, such as MASCOT or PEPTIDENT.
• The results are listed in order of statistical significance to the stored peptide data.

 Protein Capture and Validation

• Dependant on the identity of the candidate biomarker, a protein capture protocol will be devised, based often on antibody-antigen interactions. We have also devised alternative capture protocols, such as those based on protein-nucleic acid interactions.
• Captured purified proteins can be analysed in the same way as above to confirm the peptide fragment composition, thus validating the databank analysis with additional immunochemical or ligand binding data.

Protein Interaction Detection

• Immobilisation or capture of the biomarker from solution allows the analysis of interacting protein partners in the MS equivalent of the two-hybrid system.
• Multiple proteins interacting or pulled down with the identified biomarker are profiled on Ciphergen ProteinChip arrays. Differential profiles or time point assays can be analysed in this way.
• Identification of the protein partners can be performed as described above under protein identification.

Protein Affinity Measurements

• We offer analysis of association-dissociation kinetics of the required antibody-protein, protein-protein, protein-DNA or protein-ligand partners by Surface Plasmon Resonance (SPR).
• We are working with a new multiplex SPR imaging device (Genoptics Interactor^TM) that allows protein array design without the need for protein labeling using multiple parallel SPR analyses.

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