Disease Proteomics
Proteomics provides clues on complex biological and environmental factors affecting gene products.
Complex mutigenic disorders which are not necessarily inherited are:
-
cancer
-
central nervous system disorders
-
cardiovascular
-
diabetes NCBI genetic disorders
-
hypertension
Genetic as well as non-genetic diseases modify target proteins and signaling pathways.
Proteomic analysis of diseases involves the following:
-
(1) Sample acquisition: of human tissues or cells as by laser-capture microdissection (LCM) which is a method for procuring pure cells from specific microscopic regions of tissue sections. LCM uses a microscope to identify cells of interest, a special transfer film applied to the tissue section and a pulsed laser beam which melts and fuses the film with the underlying cells of choice. The film can then be lifted to extract the cells.
-
(2) Sample preparation: includes subcellular fractionation and removal of interfering DNA, RNA, lipids and carbohydrates.
-
(3) Sample separation: involves methods like 2-dimensional gel electrophoresis and liquid chromatography. Sample separation can also involve protein microarrays. In these arrays, a sample (labeled protein) is incubated with an array which is immobilized with antibodies, receptors, ligands, nucleic acids etc). Another method is the reverse-phage protein array where protein lysates prepared form cultured cells or by LCM are arrayed into nitro-cellulose slides which are then probed with antibody. These slides are then developed by colorimetric, fluorescent, or chemiluminescence. This method has been very important in studying signaling pathways.
-
(4) Protein sequence determination: involves procedures like mass spectroscopy (MS). There are 3 types: (1) MALDI (matrix-assisted laser desorption ionisation) MS, (2) electron-spray ionization (ESI-MS) and (3) SELDI/TOF (surface-enhanced laser desorption ionisation/time-of-flight) MS. SELDI protein chips are a newer method. They contain arrays of chromatographic substances with different properties such as hydrophobic, cation/anion exchange, metal affinity or biomedical interactions like antigen/antibody, receptor/ligand or DNA-protein. These chips retain the interacting molecules from the protein lysate. These chips are then laser desorbed and ionized for analysis by MS. Bioinformatics software can then be used to compare spectra from healthy and diseased patients and discriminate peak patterns. It is very useful in cancer but in slow progressing diseases subtle changes in peaks are not discriminated. These chips can be ordered from Ciphergen.
-
(5) Protein identification: involves collection of peptides from sequence-specific proteolysis. These peptide masses constitute mass fingerprints characteristic for each protein. See Bioinformatics for mass fingerprinting search engines Peptide mass fingerprints are sufficient for identification of proteins from organisms with completely sequenced genomes using programs like Peptide-Search, MS-FIT, MS-TAG.
Development of Biomarkers
Protein biomarkers are powerful descriptors of phenotype. Biomarkers may be expressed as single markers or multiple markers whose patterns are up and down regulation may signal disease. Development of new biomarkers for diagnosis at early stage is critical for treatment of cancer which spreads rapidly. The wave of the future in such analysis is by SELDI-TOF MS. An outline of this procedure is below:
Normal Serum Prostrate Cancer Serum
SELDI-TOF-MS
SELDI-TOF-MS
Comparison and Analysis
Diagnosis
Treatment and Prognosis
The above procedure can also be done with 2-D Electrophoresis, but in such a case one must extract proteins in the gel or mass spectrometry. One can also label the tumor cells and normal cells with 2 different dyes (like Cy5 and Cy3) and run those mixtures on a 2-D gel for comparison.
What does the future look like in disease proteomics? Well someday it may be possible to collect DNA from a patient, determine proteins involved in the pathological process, do a computational analysis of protein function and determination of abnormality and then design small, specific interaction molecules followed by analysis of the compound for drug interaction and toxicity which would then be tested in animals.