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With advances in proteomic approach, the discovery of disease biomarkers becomes a reality. Disease biomarkers are biological fluids which are used to examine the biological state of an individual. Biological fluids such as plasma, serum, urine and cerebrospinal fluid might contain plausible disease biomarkers. However, blood is always the main contributor to disease biomarkers. Blood is easily obtained and it can represent the biological state as blood circulates in our body system. Disease biomarkers are especially important during the early stage diagnosis of disease when treatment got its maximal effect. The uses of disease biomarkers are diagnosis of disease, differentiate the state of disease, selection of suitable treatment, monitor response to treatment as well as side effects and generate personalized medicine. The desired characteristics of good disease biomarkers are measurable so that the level of disease biomarkers can be quantitate, robust under different conditions, as well as high specificity and sensitivity to prevent false diagnosis. The challenges to discover disease biomarkers are the complex nature of proteins and the different extent of variation in protein concentration. A variety of proteins in many different concentrations are present in biological fluids, thus more sophisticated technologies in proteomic are needed to discover disease biomarkers.
(ii) Proteomics techniques used in discovery of disease biomarkers
The process in establishing useful disease biomarkers using proteomic approaches involved a few fundamentals, firstly, preparation of sample, then separation of protein using 2-dimensional gel electrophoresis (2-DGE), liquid chromatography (LC) or "protein chips". Following that is the identification of protein which can be done using mass chromatography (MS), liquid chromatography (LC) and high-resolution tandem mass spectrometry (MS/MS).
Proteomics techniques usually have better performance when in combination. For example, 2-DGE pair with MS, LC pair with MS/MS and SELDI pair with TOF-MS. 2-DGE involved the charge and size of proteins during separation. Isoelectric focusing (IEF) separate proteins according to the charge of proteins. Polyacrylamide gel electrophoresis separate proteins according to their molecular mass. After that, staining was carried out. Some examples of staining are silver staining and coomassie blue staining. Protein spots can be detected by using MS. The benefits of 2-DGE-MS are high resolving power, quantification can be carried out by using different dyes, therefore increasing the sensitivity of analysis. Moreover, 2-DGE is capable to resolve isoforms, thus useful for proteins that undergo post-translational modifications (PTMs). However, 2-DGE is not suitable for proteins with size less than 10kDa.
Matrix-assisted laser desorption/ionization (MALDI) and surfaced-enhanced laser desorption/ionization (SELDI) are two ionization method often used to analyze complex protein. MALDI ionized proteins from a dry and crystalline stage. The 3 steps involving in MALDI are firstly ionization of protein into ions (gaseous state), then ions are separated according to mass to charge ratio and lastly, detection of ions. SELDI is affinity based as chemically modified surface is used for selective binding of proteins. Buffer is used to wash off impurities. A mass spectrum with a series of spiked peaks was produced indicating the identity of protein samples according to peaks size and distance between each peak.
MALDI-TOF involved the mixture of sample with organic compounds which will then form a matrix. Laser pulse was used to vaporize peptides which are then accelerated and sent to a flight tube. The TOF to the detector is proportional to m/z for a fixed voltage. The mass was compared with theoretical mass of peptides for identification. MALDI-TOF-MS and SELDI-TOF-MS are powerful analysis method to detect disease biomarkers. SELDI-TOF-MS provides high accuracy and sensitivity in detecting complex protein samples. However, it is limited to selected proteins and low efficiency for high molecular weight proteins.
Quantitative proteomics is another important technique in the discovery of disease biomarkers. Examples of quantitative proteomics are isotope coded affinity tags (ICAT) and isobaric tags for relative and absolute quantitation (iTRAQ) which are reported in Shi et al. 2009. Isotopic light or heavy reagent has been employed to label the cysteinyl residues in reduced protein samples. Therefore, generating mass signatures that can define the origin of sample and also to quantitate the amount. However, the disadvantages is that only two protein samples can be compare at one time and it only ICAT only target the cysteine group of protein thus cannot be used for proteins that doesn't contain cysteine. While iTRAQ tagged the N-terminus of peptides. The advantages of iTRAQ over ICAT is that 8 protein samples can be analysed at the same time.
(iii) Disease biomarkers in cancer
Prostate-specific antigen (PSA) was found about 25 years ago and PSA is commonly used as a biomarker to detect prostate cancer. PSA was expressed in prostate tissue and secreted into the blood. Blood plasma level of PSA in healthy person is 2ng/mL. Increased levels of PSA can be observed in prostate cancer patient. However, PSA is not specific for prostate cancer as other factors such as age and other prostatic conditions can trigger the increase of PSA level in blood. Despite these disadvantages, PSA is useful as the mortality rate of people with prostate cancer decreases due to the early diagnosis using PSA.
Another highly used cancer biomarker is the Her2/neu proto-oncogene (CD340). CD340 was discovered in year 1980s and known to be a cancer-causing oncogenes. CD340 was first reported to cause breast cancer in rats. After further investigation, it was reported that CD340 plasma levels increased in invasive breast cancers. Normal concentration of CD340 is 10ng/mL. CD340 was secreted into the blood stream from cancer tissue and therefore can be used as a cancer biomarker.
Disease biomarkers in cardiovascular disease
There are 3 biomarkers widely used in the diagnosis of cardiovascular disease namely cardiac troponin I and T, B-type natriuretic peptides (BNP) and C-reactive protein (CRP).
Cardiac troponin I and T is a biomarker used for diagnosis of acute myocardial infarction (AMI). Cardiac troponin releases into blood stream when ischemia (restriction of blood supply in damage tissue) happened. This biomarker is specific only in myocardial tissue thus make it reliable to test for AMI. When cardiac troponin was first discovered, it has low sensitivity to detect AMI. With advances in proteomic techniques, the sensitivity of cardiac troponin increased up to 50-fold than old method. Extremely low levels can be detected in AMI and also cardiac injury such as unstable angina.
B-type natriuretic peptides can be used to diagnose chronic and acute heart failure. The B-type natriuretic peptides are specific only in ventricular tissues. This biomarker is reported to be related with left ventricular dysfunction and increased cardiovascular risk. CRP is expressed in diseased atherosclerotic arteries. Besides diagnosis purpose, CRP can be used to predict cardiovascular risk of patients.
Disease biomarkers in diabetes
Diabetes mellitus is caused by the deficiency of insulin produced. Proteins can be up-regulated or down-regulated in diabetes mellitus. The level of clusterin and apolipoprotein J increased significantly in diabetes type 2. Acetone can be used as a biomarker to detect diabetes. Acetone is reported to be increase in diabetic patients (Dong et al., 2006). The source of acetone is blood and acetone is derived from acetoacetate and isopropanol. The concentration of acetone in blood is twice the amount in healthy patient. Chromatography technique is the best tool for quantification of acetone in blood. In this study, GC-MS is used to determine acetone in human blood. Acetone can be detected in diabetes patient within a short time.
Fasting plasma glucose (FPG) is a useful biomarker to detect diabetes mellitus. World Health Organization reported that FPG can be used as diabetes biomarker in year 1998. FPG level above 126mg/dl can be concluded as diabetic (Motta et al., 2006). FPG allow early diagnosis and prevention of diabetes.
Li et al. (2008) reported 5 potential biomarkers in detecting diabetes mellitus namely glucose,Â 2-hydroxyisobutyric acid,Â linoleic acid, palmitic acid andÂ phosphate. These 5 diabetes biomarkers were discovered using two-dimensional gas chromatography-of-flight mass spectrometry (GCxGC-TOFMS). The combination of mass spectrometry and chromatography enable the detection of metabolites with high sensitivity. In this study, it reported that all these biomarkers are related with hyperglycemia and the abnormal regulation of fatty acids metabolism in diabetic patient.
Disease biomarkers in neurological disorders
The novel disease biomarkers in neurological disorders are AD and PD biomarkers. It is reported that AD biomarkers are responsible in Alzheimer's disease while PD biomarkers are responsible in Parkinson's disease (Shi et al., 2008). The source to obtain AD and PD is from cerebrospinal fluid (CSF) which are trusted to be most reliable source as it has direct contact with central nervous system (CNS). 2-DGE-, LC-, and SELDI-based proteomics were used to detect AD in human CSF. 2-DGE is sensitive and specific which successfully discriminate AD from non-AD. PD is related to the loss of neurons in the brainstem thus lead to Parkinson disease.
Amyotriphic lateral sclerosis (ALS) is a neurological disorder due to the degeneration of motor neurons. Cystatin C can be used as a biomarker to detect ALS. Cystatin C is specific for Bunina bodies, a characteristic feature of ALS. Cystatin C can be found in cerebrospinal fluid. Proteomic analysis of cystatin C proved that it is useful and specific to detect ALS (Akimoto et al., 2009).