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Biological markers, or biomarkers, are specific proteins in which their states or level of expressions are used to indicate the biological state of a person. They are often used in the prognosis or diagnosis of diseases and the effectiveness of treatments can also be monitored by biomarkers. They play a role in drug development as well, providing information on drug safety, effectiveness and individual response to drugs.
Discovery of biomarkers can be done by comparing protein maps (using methods such as SELDI) of a healthy and diseased individual. The development of specific biomarkers has to undergo various phases, including identification of promising protein candidates, qualification and rounds of validation. Biomarkers should not only be specific, sensitive and stable, they should also be useful in differentiating a diseased individual from a healthy one. A robust detection assay should be established for the biomarker as well.
Proteomics techniques used
Various proteomic techniques are applied in the discovery of disease biomarkers. They include 1D-PAGE, 2D-PAGE, Mass Spectrometry (MS), Matrix Associated Laser Desorption/Ionisation (MALDI), Surface-Enhanced Laser Desorption/Ionisation (SELDI), High Performance Liquid Chromatography (HPLC), and Microarray. Some of these techniques will be further discussed in this report.
1D-PAGE uses a reducing polyacrylamide gel to separate proteins according to their molecular weights. It involves 2 gels, stacking and resolving, which is used to focus and resolve proteins respectively. Prior to gel electrophoresis, protein samples are first suspended in a buffer containing a detergent to solubilize proteins, a reductant to break disulphide bonds, protease inhibitors, glycerol to increase density of sample for loading into wells and a tracking dye for visualization. When a charge is applied, the protein mixtures are separated into individual bands of different molecular weights. The respective molecular weights can then be estimated using a ladder.
In 2D-PAGE, proteins are first separated by their respective isoelectric points (pI) using isoelectric focusing (IEF). A pH gradient is first set up onto a gel, and the proteins will move along the gel, focusing at their pI, where they have no net charge. An IPG strip is commonly used for this. Then, the gel strip containing the samples are equilibrated in a buffer with SDS to give a overall negative change to all proteins, DTT (1st equilibration) and IAA (2nd equilibration) as a reductant to prevent the reformation of disulphide bonds, urea to denature proteins, glycerol to aid in the transfer of proteins, and bromophenol blue as a tracking dye. After that, the gel strip after equilibration is transferred onto a SDS-PAGE to separate proteins according to their molecular weights. Silver or coomassie staining is then applied for visualization of proteins.
Microarray-based proteomics is an affinity based technique which can be done in two ways - forward phase or reverse phase. For forward phase mircroarray, a nitrocellulose-coated slide is used to immobilize monoclonal antibodies. Various protein samples are then directly labeled with different fluorophores are applied to the array. Different fluorescent signals are detected and recorded when binding occurs, making this a sensitive method capable of analyzing low-abundance protein. This allows comparison of expressions between various samples, and is especially useful when many proteins are needed to be measured simultaneously. Alternatively, an indirect sandwich assay can be used, involving secondary antibodies. In this case, the secondary detection antibodies are the ones being labeled with fluorophores instead of the samples. For reverse phase microarray, sample mixtures are first immobilized onto a solid substrate before specific antibodies are added for screening. These antibodies are labeled with fluorophores to allow detection of the presence of target proteins. However, some shortcomings of this technique are that identifications are restricted to the available antibodies used and the probabilities of antibody cross-reactivity.
Surface-enhanced laser desorption/ionization (SELDI)
SELDI is an affinity based MS method which involves the binding of proteins from complex samples to different arrays. The array surfaces used include hydrophobic surfaces, metal-binding surfaces, cation and anion exchange. Specific bindings onto these arrays are dependent on the biochemical properties of different proteins and their affinity for each array. Unbound proteins are then removed by washing with a buffer, before the bound proteins are subjected to TOF-MS for quantification and mass measurement. This technique allows fast comparative analysis of different samples and biomarker discovery can be done by comparing the spectra obtained from diseased patients and healthy individuals.
Cancer biomarkers are generally categorized into three groups - diagnostic, prognostic and predictive. Examples of diagnostic markers are PGP 9.5 (a neurospecific protein for lung cancer) and RS/DJ-1 (RNA-protein interaction regulator) for breast cancer. As for prognostic and predictive biomarkers, they include FK506BP (regulatory protein) and glyoxylase I (for detoxification) for ovarian cancers respectively.
A potential biomarker ribosomal P2 which predicts the sensitivity to a PI3K inhibitor used in cancer therapy was also discovered by Akashi T. et al.,(2007). The experiment involved the study of 393 proteins in 39 human cancer cell lines expression using SELDI-TOF-MS and the degree of phosphorylation of ribosomal P2 was found to correlate with to LY294002 sensitivity. Discovery of such biomarkers which predict the chemosensitivity to cancer therapy is important in studying the effectiveness of therapies and prevent any side effects.
Cardiovascular Disease Biomarkers
Cardiovascular diseases are prevalent in many developed countries. Some biomarkers of cardiovascular diseases include, Cardiac Troponin-I/T, LD (LDH), serum amyloid A protein, Creatin Kinase (CK/CK-MB), hemoglobin and C-reactive protein.
An example to be further elaborated is the cardiac Troponins (cTnI and cTnT), which are uniquely expressed by heart muscles. They usually not found in normal individual's serum and detection of elevated levels in the blood may imply damage to cardiac muscles, cardiac chronic disease or acute myocardial infarction (AMI). In cases of AMI, elevated levels of TnI and TnT levels will remain even after ten days and hence is good for retrospectively diagnosing AMI.
A heat shock protein, HSP27, was identified as a possible novel biomarker as well. In healthy individuals, HSP27 production is significantly higher than in carotid atherosclerosis patients. This implies that reduction in HPS27 levels can be used as a biomarker for atherosclerosis disease. However, HSP27 still needs to be tested on a larger group of individuals in order to be validated. (Martín-Ventura J.L. et al., 2004)
Diabetes mellitus is a condition whereby an individual suffers from hyperglycemia, either due to a lack of insulin (Type 1) or insulin resistance (Type 2) or both. Currently, the Oral Glucose Tolerance Test (OGTT) and Fasting Plasma Glucose (FPG) are used to determine diabetes risk, but they each have their shortcomings. For instance, OGTT requires two hours to be done and poses inconvenience for the patient, while FPG exhibits poor specificity. (Gedela S. et al., 2007)
In a study done by Riaz et al., (2009), they had identified four potential novel biomarkers for Type 2 diabetes - Apolipoprotein A-I, Apolipoprotein E, C-reactive protein(CRP) and Leptin. Samples were collected from 125 diabetic patients and 50 healthy individuals as control for this experiment. By using Bradford assay, the total serum and urinary proteins were measured while unwanted proteins, such as albumin and immunoglobulins, were extracted. The samples were then analyzed using chromatofocusing and reverse-phase HPLC, while the three target proteins are identified using MS. These proteins were then quantified using ELISA and results are compared between the diabetics and control group. It was found that the levels of Apolipoprotein E, CRP and Leptin were highly elevated in the diabetics while Apolipoprotein level was down-regulated.
Neurological Disorders Biomarkers
An example of a neurological disorder is Alzheimer's disease (AD), which is an incurable neurodegenerative disorder. Being the most prevalent form of dementia, development of a sensitive, specific and reliable biomarker for it is crucial as it can aid the prediction and diagnosis of the disease. Currently, some biomarkers of AD include decreased levels of Beta-amyloid and increased levels of Tau protein measured in CSF.
In an experiment by Carrette O. et al., the change in expression levels of 5 proteins in the CSF of 9 AD patients and 10 healthy individuals was detected by SELDI ProteinChip array. These proteins are reflected as Cystatin-C, a 4.8 kDa VGF polypeptide, two h-2-microglobulin isoforms, 4.8 kDa VGF polypeptide, and an unknown 7.7 kDa polypeptide by using mass spectrometry. However, to use these proteins are valid biomarkers still needs to be speculated.