Differential Protein Profiling
LEAP technology was further explored for its application on differential protein profiling for biomarker discovery using patient samples. Serum proteins from four multiple myeloma patients and a normal control were first labeled with biotin before subjecting them to a LEAP ligand-enrichment method using NIH3T3 cells as the receptor carrier.
The enriched ligands were then run on 1-D SDS PAGE followed by a Western blot. The biotin-labeled ligand proteins were detected by HRP-conjugated streptavidin. The profiles of the multiple myeloma patients share an elevated level of a protein migrated at position X, suggesting protein X as a biomarker candidate for multiple myeloma (Figure 3).
Advantages of the LEAP technology include selective enrichment of biologically functional (based on binding) ligands specific to desired targets; simple elimination of unwanted proteins; enriched samples that can be subsequently analyzed using conventional methods—2-D DIGE, Western blotting, LC/MS, etc.; the ability to profile both known and unknown ligands; and applicability to any ligand/receptor association and interaction of any two interacting molecules that are polypeptides or nonpolypeptides.
LEAP technology has broad applications in the discovery of novel ligands for diagnostic and therapeutic purposes, assessment of therapeutic potential in nonclinical studies, and the monitoring of patient response to a specific treatment regimen.
Stem cell research is currently in need of accurate markers to distinguish stem cells of different origin such as embryonic stem cells and adult stem cells. LEAP technology can provide a solution for accurate typing of stem cells of different origins by generating a characteristic ligand profile for stem cells of each origin using a universal ligand mixture. Since each derived ligand profile reflects the receptor profile of the corresponding stem cells, the ligand profile should serve as a fingerprint for each type of stem cell.
Biomarkers for plaque rupture would be useful in predicting heart attack and stroke. Plaque rupture is initiated by the break of the fibrous cap, which is composed of smooth muscle cells (SMC). LEAP technology can be applied to discover biomarkers associated with plaque rupture by comparing ligand profiles of SMC from the sera of the same patient collected before and after heart attack.
LEAP technology can also be applied to identify satiety molecules through differential protein profiling of ligands enriched from sera of the same individual collected at hungry and full states using hypothalamus neurons as the receptor carrier.
To date, there are more than 100 forms of arthritis currently affecting one in five adults in the U.S. The characterization of ligand profiles of synovial fluid from patients with various types of arthritis may provide biologists information for designing new therapeutics for arthritis and also with biomarkers for accurate diagnosis.
LEAP technology can also be applied to discover unidentified ligands for orphan receptors through differential protein profiling of ligands enriched from a ligand-containing mixture using both orphan receptor null cells and orphan receptor expressing cells derived from the null cells.
The simplicity, efficiency, and biological relevance of the LEAP technology should improve the odds for biomarker discovery in diagnostic, therapeutic, and basic research areas. First-generation products are currently in beta testing and will soon be available to all researchers in the biomedical community.