The Critical Need

It is well established that, for a wide range of diseases, a particular drug’s clinical benefit during treatment varies widely within patient populations.  The discovery and validation of gene and protein biomarkers that classify patients into sub-populations for a more “personalized” approach to therapy have thus emerged as a critical part of drug development, disease diagnosis and management.  The enormous heterogeneity of patients, however, presents a very difficult challenge to the general use of comprehensive protein profiling in a clinical setting.  New approaches are clearly needed to dig deeper the phenotype of disease onset and progression and to better understand how to develop and monitor improved therapies for patients. 

Eprogen’s “Bedside-to-Bench” Solution

Eprogen’s solution to this is based on the established notion that a patient’s immune response can serve as a sentinel for over- or under-expression of groups of proteins which form a unique protein “profile” or signature of a particular disease. To comprehensively interrogate the patient immune response, a reference cell line or tissue sample specific to a disease is prepared and 2D fractionated (in the liquid phase) to “array” the proteins present into well-defined subsets for probing. 

These protein fractions from the reference samples are then “spotted” onto numerous glass slides to produce identical protein microarrays.  This process significantly reduces the complexity of the reference sample and allows for simultaneous probing of 1000’s of distinct protein fractions in one easy step.  Thousands of these disease specific reference microarrays can be produced from one 2D fractionation of the reference sample.  In addition, the individual liquid fractions used to prepare the microarray spots are stored for later access and in-depth analysis of the proteins they contain. 

Using defined cohorts of patient sera, serological assays are performed on each patient sample (or pooled samples) using these reference microarrays.  Through statistical analysis of the reactive protein “spots” in these microarrays, typically measured using fluorescence, autoantibody signatures are established for the particular disease reference sample.  This then identifies the “biologically relevant” protein spots that classify patients in the cohorts into respective populations or subpopulations.  Using normal control patients along with benign or non-related disease patients, the same serum samples can be screened across many different reference sample microarrays to generate a global patient profile with relative ease.

Once the protein fractions (spots) that differentiate the patient populations of interest are identified (typically < 5% of the total number of spots on the microarray) the liquid fraction used to prepare the spot on the microarray can be readily accessed for detailed analysis of the proteins present for validation and further research.   This approach in reality “prioritizes” the spots containing the proteins of interest.   Now, biologically important candidate biomarkers are identified before resorting to more sophisticated techniques (like MS) to characterize the exact nature of the proteins expressed.  Having a good clinical basis at the outset to pursue a protein or group of proteins as biologically significant is of great value.

These characteristic auto-antibody profiles serve not only to highlight active biological pathways related to the disease onset or progression, but also help to “discover what we do not know” about these active pathways as well.  These immune response profiles of patient samples (Bedside) are now used to generate new, or improve on existing strategies of drug and biomarker development (Bench) by first discerning the relevant proteins (biomarkers) that are truly associated with the disease onset and progression.

The unique aspects this “Bedside-to-Bench” approach offers and the efficiencies this will introduce in both time and cost will have a significant impact on how protein biomarker and drug discovery research as well as clinical trials are performed in the future.   If the same process can also be used for both drug development efforts and to monitor patients before, during and after the drug treatment, it would save enormous amounts of time, effort and money in drug and biomarker development and validation costs.  It would also serve as a significant aid to clinicians dealing with patients throughout the course of diagnosis and treatment.

ProteoSep Microarray Strategies:  Make More Effective Use of Clinical Samples!

Cell Line or Tissue Screening/Profiling

Western Blot alternative:

• 1D & 2D liquid multi-well fractions contain intact proteins for direct probing with antibodies.
• Screening multiple arrays produced from the same sample with many different antibodies!
• Antibody qualification and coverage assays.

PTM analyses – Phospho-, glyco-protein detection and coverage.

Serological (biofluid) assays:

• Drug treated vs. Control studies with patients – clinical trial monitoring

• Autoantibody signatures or profiles – Patient (sub)classification

• Disease progression analysis

• New Biomarker discovery studies

References:

• 1D Glyco Protein Arrays with Multi-Lectin Detection

Qiu Y, Patwa TH, Xu L, Shedden K, Misek DE, Tuck M, Jin G, Ruffin MT, Turgeon DK, Synal S, Bresalier R, Marcon N, Brenner DE, Lubman DM.

Plasma Glycoprotein Profiling for Colorectal Cancer Biomarker Identification by Lectin Glycoarray and Lectin Blot.

J Proteome Res. 2008 Apr 4;7(4):1693-1703.

• 2D Phosphorylation and Glycosylation Mapping Microarrays

Pal M, Moffa A, Sreekumar A, Ethier SP, Barder TJ, Chinnaiyan A, Lubman DM.

Differential phosphoprotein mapping in cancer cells using protein microarrays produced from 2-D liquid fractionation.

Anal Chem. 2006 Feb 1;78(3):702-10.

Zhao J, Patwa TH, Pal M, Qiu W, Lubman DM

Analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry.   

Methods in Mol Bio., 2009;492:321-51.

• 2D Humoral Response Cancer Lysate Arrays

Taylor BS, Pal M, Yu J, Laxman B, Kalyana-Sundaram S, Zhao R, Menon A, Wei JT, Nesvizhskii AI, Ghosh D, Omenn GS, Lubman DM, Chinnaiyan AM, Sreekumar A.

Humoral response profiling reveals pathways to prostate cancer progression.  Mol Cell Proteomics. 2008 Mar;7(3):600-11.