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Proteomics: an evolving technology in laboratory medicineInternet Journal of Medical Update 2010 January;5(1):35-41 Internet Journal of Medical Update
Journal home page: http://www.akspublication.com/ijmu
Proteomics: an evolving technology in Laboratory Medicine
Dr. Javed Akhter*Ψ PhD, Dr. Waleed Al Tamimi* PhD, Dr. Abubaker El Fatih*
FRCPath and Dr. D J Venter† MD
*Department of Pathology and Laboratory Medicine, King Abdulaziz Medical
City, Riyadh, Saudi Arabia
†Department of Pathology and Laboratory Medicine, Mater Health, Brisbane,
(Received 23 February 2009 and accepted 12 April 2009) ABSTRACT: The rapid developments in both genomics and proteomics
will allow scientists to define the molecular pathways in normal and
diseased cells. With these models, researchers will have the ability to
predict previously unknown interactions and verify such predictions
experimentally. Novel proteins, cellular functions, and pathways will also
be unravelled. It is hoped that understanding the connections between
cellular pathways and the ability to identify their associated biomarkers
will greatly reduce the suffering and loss of life due to diseases.
KEY WORDS: Proteomics; Laboratory medicine
different proteins. On average, however, a gene produces five to ten different proteins4.
Genomics is the systematic use of information was a pivotal first step to revolutionize on the expression, regulation and structural medicine in the 21st century1. The completed
association of genes. It is used in genetic human genome was found to contain between analysis, measurement of gene expression and 30,000 and 35,000 genes, far less than the determination of gene function5. As genomics
100,000 genes predicted when the project has proven inadequate to predict the structure commenced in the mid-1990s2. Subsequently it
and dynamic properties of all proteins, a new was found that one gene can produce more field of protein study termed proteomics has developed. This is the large-scale study of functional capability. The generation of protein expression, structure and function. It multiple proteins from a single gene can occur aims to correlate the structural and functional as a result of alternate splicing where a single diversity of proteins with underlying biological DNA template can produce several different processes, including disease processes6.
messenger RNAs, each of which is then used to make different proteins3. In addition, the
identify, investigate and target proteins that are protein may undergo modification by cellular differentially expressed in health and disease. processes after it is created (termed post- translational modification). The result is that enormously with the potential to develop better diagnostic and prognostic tests, to identify new therapeutic targets and ultimately to allow patient-individualized therapy. Finding the protein or proteins (biomarker) associated with ΨCorrespondence at: Department of
a disease or adverse event will lead to a much earlier identification of disease, potentially Abdulaziz Medical City, Riyadh 11426, Saudi prior to the onset of symptoms7.
Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine TECHNOLOGIES
scientific challenge to amplify precisely. The first high resolution protein separations Hence, proteins are studied using a synergistic electrophoresis in 1975; this was followed by the first computerised 2-D gel image analysis chromatography and bioinformatics11.
platform to quantify changes in 2-D gel protein 2D gel electrophoresis is a suitable technique spot levels8. However there was a lack of
for asking, “Where do differences arise useful tools to identify proteins of interest. amongst the proteins in two similar samples?” Furthermore, the lack of reproducibility For example, closely matched samples from hindered the expansion of the technique until diseased and healthy cells can be compared. the introduction of immobilized pH gradients Differences in protein abundance or covalent (IPGs) in the 80s. This has coincided with the development of mass spectrometry ionization glycosylation and acylation) can provide techniques for peptides, allowing protein important clues to the pathogenesis, progress identification and characterization on a large and treatment of a disease. Once a protein has scale9. However, it was not until the mid-90s
that mass spectrometry became a mainstream technique for protein identification, mostly suitable tool for asking, “What is this replacing Edman sequencing10.
protein?”, “Which residues are modified?”, Currently, there is no diagnostic amplification and “What is the modification?” By taking 3-D pictures of proteins, X-ray Tomography allows amplifying genes. It is therefore not possible to researchers to see biomolecules in their cellular make copies of proteins that are present in very context. Tomograms provide insights into the small amounts. Another challenge is that conformation and flexibility of functional amino acids are very small, ranging from 7 to targets and their environment (Table 1).
Table 1: Technologies used in proteomics
Used to identify low abundance proteins in complex biological samples such as blood, urine and oral fluid. Used to separate ions based on a sample's electronic mass, to study inborn errors of metabolism and metabolic profiles, and to identify therapeutic drugs, drugs of abuse, disease markers and toxic compounds. Mass spectrometry MALDI-TOF (Matrix Deals with thermolabile, non-volatile organic Assisted Laser Desorption Ionisation-Time compounds and those of high molecular mass. It is used Of Flight) in for the analysis of proteins, peptides, glycoproteins, oligosaccharides and oligonucleotides. ICP-MS (Inductively Coupled Plasma- Involves the formation of gas containing electrons, ions Mass Spectrometry ) and neutral particles from Argon gas. Technology is used for ultrasensitive quantification of proteins and peptides down to low attomole range. Used to determine the location of labelled proteins or protein complexes in an intact cell. Frequently correlated with images of cells from light based microscopes. These are matrix-support surfaces for binding selected proteins and allowing high-throughput screening for disease associated proteins. These methods are used for detection of drug-protein, hormone-protein, protein-protein, DNA-protein, carbohydrate-protein, and lipid-protein interactions. Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine CLINICAL APPLICATIONS
nearly two million people each year. More than 50% of TB cases occur in the largest Asian The potential applications of proteomics in the Bangladesh, Philippines and Pakistan). Sub- components of the proteome; comparing the Saharan Africa has the highest incidence rate expression of proteins between normal and (approximately 300/100,000 population/year). diseased organs at certain stages of disease; bioinformational analysis to determine how Western Europe and North America, the global proteins interact with each other in vivo; TB burden appears on the rise, especially in identification and characterization of proteins the former Soviet Union, Eastern Europe, and post-translationally; study the structure and Africa15.
function of protein complexes to understand A serum or saliva-based screening test that the organization of cells at the molecular level. could detect pre-clinical infection would allow The goal of clinical proteomics and molecular transmission, and have widespread application. characterization of the cellular components and Proteomic techniques have identified proteins secreted in vitro by common clinical isolates. understanding of the pathology of disease process, diagnosis and patient management. The translational nature of this technology antigens, with sensitivity of 60%–74% and provides unique challenges and boundless specificity of 96%–97% in clinical studies. opportunities that promise to transform the These proteins are potential candidates for a managed12. It has many clinical applications
Diagnosis of Severe Acute Respiratory
• Translational pathology and Syndrome: The pathogenesis of severe acute
understood, and a specific diagnostic method is • Bioinformatics tools including pattern critical for the management and control of this recognition, artificial intelligence and disease. Proteomic analysis of sera from patients with SARS has identified potential • Biomarker discovery and validation from biomarkers. These are truncated forms of α (1)-Antitrypsin, which were consistently found • Signal transduction pathways profiling in in higher concentrations in the sera of SARS patients compared with healthy controls. These markers may prove useful as diagnostic tools and therapeutic targets. Moreover, studies of the protein structure of the SARS virus may Use of proteomic technologies in the drug development pipeline reveal potential vaccine targets16.
Use of proteomic technologies to monitor prognosis, therapeutic end points, toxicity PROTEOMIC AND CANCER
Many studies using proteomic techniques have Clinical trials using proteomic monitoring been performed on biomarkers to investigate Some of the major areas in which clinical potentials of early cancer diagnosis17.
cardiovascular disease, Alzheimer's disease, Ovarian Cancer: Ovarian cancer represents
infectious diseases, infertility, obstetrics and the sixth most commonly diagnosed cancer immune rejection following transplantation13.
among women in the world, and causes more deaths per year than any other cancer of the TARGETED MODALITIES OF female reproductive system. Ovarian cancer is
North American countries. Ovarian cancer is a major focus of early biomarker discovery Diagnosis of infectious diseases: Tuberculosis
because it is usually diagnosed at an advanced (TB) affects millions of people around the stage with a median five-year survival rate of about 20 percent18. To evaluate the potential
Mycobacterium tuberculosis strains spreading use of proteomics as a diagnostic tool, a group worldwide14. Among the communicable
diseases, TB is the second leading cause of Institute (NCI) in Bethesda, MD, collected Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine serum from 50 ovarian cancer patients and 50 cancer harbour microscopic metastasis at the controls and used a computer algorithm to time of diagnosis. It is now well established distinguished cancer cells from non-cancer survival in patients with early-stage breast cells. They have shown that with a set of cancer. Recent technical advances in mass spectrometry, such as matrix-assisted laser correctly identified all 50 patients with cancer, desorption/ionisation time-of-flight mass and was able to discriminate them from 63 out of 66 patients without cancer or had benign disease. Using the same approach, two other desorption/ionisation time-of-flight mass groups reported similar results19,20.
spectrometry (SELDI-TOF MS), have enabled high-throughput proteome analysis26,27.
Prostate cancer: The worldwide incidence of
A multitude of molecules involved in breast cancer biology have been studied as potential cancers in men. The highest incidence of prostate cancer in the world is found in combination of three candidate proteins in the American black men, who have approximately a 9.8% lifetime risk of developing this cancer discriminating between 169 patients at various compared to the 8% lifetime risk for American stages of breast cancer compared to women Chinese populations have the lowest rates of controls28. In other studies, nipple aspirate
prostate cancer21. Since the advent of prostate
specific antigen (PSA) screening, a significant candidates29. Proteomic analysis of breast
number of men have had a PSA test performed nipple aspirate fluid (NAF) holds promise as a and this has led to a significant increase in the non-invasive, low cost method to identify number of diagnosed cases22. However, the
PSA lacks sensitivity and therefore, evaluating molecules when secreted, they represent the final processed form of the marker protein, establishing signature proteomic patterns that distinguish cancer from non-cancer as well as ambiguous to provide clues to changes in identify all genetic subtypes of the cancer and protein translational rates, post-translational modification, sequestration, and degradation In one study, proteomic analysis of prostate that lead to disease. Many of the proteins that cancer patients versus healthy controls was have been identified in the NAF proteome carried out by looking for differences in could potentially be markers of disease, protein patterns between the two groups. Using including ras-related protein; metastasis- associated protein; BCL2, which has been patients, 77 patients with benign prostate implicated in the suppression of cell death; CD5, which is reported to play a role in the patterns developed as a classification system inhibition of apoptosis; retinol-binding protein, had correctly classified 96 percent of the which has recently been shown to suppress samples as either prostate cancer or non-cancer breast cancer cell survival and has been shown (benign prostate hyperplasia/healthy men)23. A
to be down-regulated in a subset of breast further proteomic approach is to determine cancer; clusterin, which has been associated with cell death and apoptosis; and transferrin, phosphoproteins believed to be involved in proliferation30.
progression in prostate cancer patients have been speculated to serve as a biomarker of Bladder Cancer: Bladder cancer incidence
varies widely throughout the world. Belgium and Italy, , have the highest recorded incidence Breast Cancer: Breast cancer is the most
rates in Europe (42.5/100,000 and 41/100,000 population respectively), much more than in Western world and constitutes 18% of all cancers in women25. Traditional prognostic
24.1/100,000 and an estimated 61,160 newly factors include the axillary lymph node status, diagnosed cases in 2007. However,, cancer the tumor size, the nuclear grade and the registries in Slovenia, Croatia, and Switzerland histologic grade. Interest in novel prognostic have reported even lower European bladder markers is based on the fact that a significant cancer incidence (10.1/100,000, 11.7/100,000 number of patients with early-stage breast and 12.0/100,000 respectively) with the lowest Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine resistance. Proteomics technologies are playing countries. Bladder cancer affects men four a major role in identifying potential therapeutic times more often than women. The risk of targets in Plasmodium species, as well as host- bladder cancer increases with age with over 70 percent of people diagnosed are older than 65 interactions. Advances to date include the years31,32.
Biological characteristics of urothelial Plasmodium species, identification of immune carcinomas range from benign, superficial, targets for vaccination and immune protection, low-grade, non-life threatening, papillary better understanding of the cellular target(s) of lesions, that respond well to resection and adjuvant treatment but are prone to recurrence chloroquine resistance36.
to highly invasive malignant carcinomas with Development of new Therapeutic Agents
demonstrated that specific protein patterns can Proteomics as an evolving science is expected be detected from tumor tissue and these could to have a major impact on drug development in discriminate adequately between diseased and the near future. It has been shown that some healthy tissue. In the case of bladder cancer, proteins which are differentially expressed by proteomics analysis has identified several microorganisms, and that differ primarily in keratin proteins that are expressed in different thier tertiary structure from related proteins in amounts as the disease progresses from the early transitional epithelium stage to full therapeutic drug targets. These can be tested against commercially available libraries of measurement of keratin levels in bladder chemical agents to identify lead compounds - cancer biopsies can therefore be used to compounds with in vitro activity that can be used to target these protein markers and to Another protein, psoriasin, is found in the represent potential new therapies. Exploitation urine of bladder cancer patients and can be of these scientific findings could assist to used as an early diagnostic marker for the disease. The study and utilization of these challenge the complexity of various clinical proteomics, but not DNA arrays, can be used in cancer diagnosis. Urine, in common with FUTURE DEVELOPMENTS
most bodily fluids, contains proteins but no RNA33,34.
At present, it is fairly premature to utilize PROTEOMICS AND THERAPEUTICS
using proteomics analyses as screening or diagnostic tools. However, these exploratory Drug Resistance
studies point to the promise of proteomics as an investigatory tool to be used to screen or Drug resistance represents a major clinical diagnose many disease entities using newly obstacle in the management of many infectious diseases, and, in many cases, the mechanism is Applied research in medical diagnostics is unknown. Genetic and protein-sequence data being developed and continues on several for many microorganisms is now available and metabolic, inherited, infectious and malignant disease entities with construction of proteomic resistance to drugs and for identifying novel agents for treating drug-resistant disease, such as azole resistance in Candida albicans which These include amniotic fluid biomarkers which has been linked with differential expression of proteins such as Erg10p, a protein involved in determination of fetal pathology biomarkers. the ergosterol biosynthesis pathway. This has been shown as a potential drug target for the biomarkers' alterations, special attention has treatment of resistant disease35.
been given to elucidation of the pathogenesis and the etiology of female infertility, and of Chloroquine resistance: Chloroquine has been
recurrent miscarriages with the elaboration of one of the most successful drugs used to treat clinical algorithms for the management of ineffective in many parts of the world by the Further research is needed to examine specific features of posttranslational modification of Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine peptide hormones that could as markers of some pathological processes such as colorectal determination of pharmacogenetic approaches 6. Liotta LA, Espina V, Mehta AI, et al. Moreover, continuous utilization of genomics biomarkers to screen and diagnose malignant 7. Petricoin EF, Ardekani AM, Hitt BA, et tumors, has lead to and continues to discover al. Use of proteomic patterns in serum to and evaluate many of the markers for the so- called "silent tumors" of bone, ovaries, proteins such as myc, src, fos, jun, myb, fms, and raf-1. This is being carried out as an aid to screening for the presence of these cancers. 9. Ahmed FE Utility of mass spectrometry construction of proteomic maps of malignant for proteome analysis: part I. Conceptual purposes, and to aid an early diagnosis and proteomics modalities and techniques is now 11. Tyers M & Mann M. From genomics to such as the diagnosis of chronic hepatitis, whereby, scientific research have continued to Hepatitis B and Hepatitis C viruses to aid in the diagnosis and the control of therapeutic efficacy37,38.
Validation of these new tests in large clinical trials is necessary prior to implementing proteomics techniques and patterns routinely in clinical use as tools for early disease detection. It is anticipated that databases of several proteins from tissues, cells and body fluids in health and disease shall soon be available. diseases. Infect Disord Drug Targets. 2008 These will link multiple parameters such as expression of specific proteins and cellular 15. Dye C, Scheele S, Dolin P, Pathania V, pathways and proliferation with genetic data and disease states that can be used for accurate Global burden of tuberculosis: estimated and rapid diagnosis39.
complete. News from The Scientist. 2. Verrills NM Clinical Proteomics: Present genome for better drug therapy. Annu Rev 3. Venter et al. The Sequence of the Human 19. Jurisicova A, Jurisica I, Kislinger T. Advances in ovarian cancer proteomics: the quest for biomarkers and improved Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine 20. Triche TJ, Schofield D, Buckley J (2001) Fluid. Clinical Cancer Research Vol. 10, in four member countries (Cyprus, Egypt, with US SEER. National Cancer Institute. descent: a comparative literature review of 32. American Cancer Society [homepage on the Internet]. Cancer facts and figures, Proteomic analysis and identification of new biomarkers and therapeutic targets for translational cancer research: toward an 34. Trevino V, Falciani F, Barrera-Saldaña cancer. World J Urol (2007) 25:557–562. genomic tool for biomedical and clinical 35. Thomas DP, Pitarch A, Monteoliva L, Gil matching algorithm distinguishes prostate pathogenicity. Infect Disord Drug Targets. 25. Watson R W G and Schalken J A Future 26. Esteva FJ and Hortobagyi GN . Prognostic molecular markers in early breast cancer Breast Cancer Res. 2004; 6(3): 109–118. 38. Holzmuller P, Grébaut P, Brizard JP, 28. Meric-Bernstam F, Serum Proteomics for
29. Sauter ER, Zhu W, Fan XJ, Wassell RP, analysis of nipple aspirate fluid to detect biologic markers of breast cancer. Br J 30. Alexander H, Stegner AL, Wagner-Mann Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved
Computerized Medical Imaging and GraphicsAtherosclerosis: The evolving role of vascular image analysisNational Technical University of Athens, School of Electrical and Computer Engineering, Biomedical Simulations and Imaging (BIOSIM) Laboratory,Iroon Polytechniou 9, Zografos 15780, Athens, GreeceAtherosclerosis, a chronic systemic degenerative disease involv-hemorrhage, large lipid cores,