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Proteomics: an evolving technology in laboratory medicine


Internet Journal of Medical Update 2010 January;5(1):35-41 Internet Journal of Medical Update
Journal home page: http://www.akspublication.com/ijmu
Review Article
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,
Australia
(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

INTRODUCTIONΨ

different proteins. On average, however, a gene produces five to ten different proteins4.
Completion of the Human Genome Project 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 than one protein, each with a different 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 Proteomics has created opportunities to 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- Clinical medicine is poised to benefit translational modification). The result is that enormously with the potential to develop better one gene can produce as many as 1,000 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 Pathology and Laboratory Medicine, King earlier identification of disease, potentially Abdulaziz Medical City, Riyadh 11426, Saudi prior to the onset of symptoms7.
Email: AkhterJ@NGHA.MED.SA Copyrighted by Dr. Arun Kumar Agnihotri. All right reserved Akhter et al/ Proteomics: an evolving technology in Laboratory Medicine 24 atoms, which represents an immense scientific challenge to amplify precisely. The first high resolution protein separations Hence, proteins are studied using a synergistic were achieved by two-dimensional gel combination of electrophoresis, mass electrophoresis in 1975; this was followed by spectrometry, multidimensional liquid 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 modification (e.g. phosphorylation, 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
been isolated and digested, the mass that mass spectrometry became a mainstream spectrometer (ICP-MS, MALDI-TOF) is a 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 technique for proteins as there is for 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
Technology Uses
2-D Gel electrophoresis Used to identify low abundance proteins in complex biological samples such as blood, urine and oral fluid. Tandem mass spectrometry 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. X-ray Tomography 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. Microarray ‘chips' 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, 1. Affinity chromatography hormone-protein, protein-protein, DNA-protein, 2. Yeast two hybrid techniques carbohydrate-protein, and lipid-protein interactions. 3. Fluorescence Resonance Energy 4. Surface Plasmon Resonance (SPR) 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 countries (India, China, Indonesia, laboratory revolve around: identifying 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; Even though TB has declined steadily in 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 early treatment, potentially reducing medicine is to assist in the study of transmission, and have widespread application. characterization of the cellular components and Proteomic techniques have identified proteins cellular networks to be used in the secreted in vitro by common clinical isolates. understanding of the pathology of disease Two of these (rRv3369 and rRv3874) have process, diagnosis and patient management. shown great potential as serodiagnostic 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 way disease is diagnosed, treated and kit-based serum screening test. managed12. It has many clinical applications
including the following: Diagnosis of Severe Acute Respiratory
• Translational pathology and Syndrome: The pathogenesis of severe acute
immunohistochemistry applied to protein respiratory syndrome (SARS) is not well biomarkers in tissue 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 computer learning algorithms patients with SARS has identified potential • Biomarker discovery and validation from biomarkers. These are truncated forms of α clinical samples (1)-Antitrypsin, which were consistently found • Signal transduction pathways profiling in in higher concentrations in the sera of SARS clinical tissue samples patients compared with healthy controls. These • Discovery of new drug targets from markers may prove useful as diagnostic tools clinical samples 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.
proteomics are utilized include cancer, 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
PROTEOMIC CLINICAL
more common in Northern European and 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 globe with many drug-resistant about 20 percent18. To evaluate the potential
Mycobacterium tuberculosis strains spreading use of proteomics as a diagnostic tool, a group worldwide14. Among the communicable
of researchers from the National Cancer diseases, TB is the second leading cause of Institute (NCI) in Bethesda, MD, collected death worldwide after HIV-AIDS, killing 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 search for the protein patterns that that adjuvant systemic therapy improves 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 blinded serum samples, the test pattern 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 spectrometry (MALDI-TOF MS) and its of 66 patients without cancer or had benign variant surface-enhanced laser 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 prostate cancer (PCa) ranks third among cancer biology have been studied as potential cancers in men. The highest incidence of prognostic markers. In one study a prostate cancer in the world is found in combination of three candidate proteins in the American black men, who have approximately blood were found to be useful in 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 white men. The Japanese and mainland with benign breast disease and healthy Chinese populations have the lowest rates of controls28. In other studies, nipple aspirate
prostate cancer21. Since the advent of prostate
fluid was used to identify tumor marker 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
markers of breast cancer. These protein PSA lacks sensitivity and therefore, evaluating molecules when secreted, they represent the multiple proteins will be essential to final processed form of the marker protein, establishing signature proteomic patterns that which makes proteomic analyses less 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 their biological activity. 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- blood samples from 167 prostate cancer associated protein; BCL2, which has been patients, 77 patients with benign prostate implicated in the suppression of cell death; hyperplasia and 82 healthy men, protein 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 whether the changes in specific with cell death and apoptosis; and transferrin, phosphoproteins believed to be involved in which has been assigned a role in cell cellular signalling events and cancer progression in prostate cancer patients have been speculated to serve as a biomarker of Bladder Cancer: Bladder cancer incidence
early disease24.
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 common malignancy among women in the population respectively), much more than in Western world and constitutes 18% of all the United States with an incidence of 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 rates found in Asian and South American 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 pathogen interactions and protein-drug percent of people diagnosed are older than 65 interactions. Advances to date include the years31,32.
identification of differences between 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 chloroquine and the mechanisms of adjuvant treatment but are prone to recurrence chloroquine resistance36.
to highly invasive malignant carcinomas with Development of new Therapeutic Agents
Several laboratories have successfully 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 the host have now become potential early transitional epithelium stage to full therapeutic drug targets. These can be tested blown squamous cell carcinoma. The 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 monitor the progression of the disease. 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 develop improved therapeutic agents to disease. The study and utilization of these challenge the complexity of various clinical novel markers support the notion that 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 many of the newly discovered biomarkers 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 discovered biomarkers. 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 provides tools for understanding their disease entities with construction of proteomic resistance to drugs and for identifying novel maps of many serum and body fluids 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 are being studied for the complex proteins such as Erg10p, a protein involved in determination of fetal pathology biomarkers. the ergosterol biosynthesis pathway. This has Moreover, based on dynamics of specific 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 malaria but has been rendered virtually these conditions ineffective in many parts of the world by the Further research is needed to examine specific widespread emergence of chloroquine 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 5. Zhu H, Snyder M. Protein arrays and some pathological processes such as colorectal microarrays. Curr Opin Chem Biol. and breast cancer, to assist with the determination of pharmacogenetic approaches 6. Liotta LA, Espina V, Mehta AI, et al. to target and manage these conditions Protein microarrays: meeting analytical Moreover, continuous utilization of genomics challenges for clinical applications. and proteomics to study and examine Cancer Cell. 2003;3:317-25. 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- identify ovarian cancer. Lancet. called "silent tumors" of bone, ovaries, pancreas and others. In particular, the 8. Issaq H, Veenstra T. Two-dimensional determination of proto-oncogene-encoded polyacrylamide gel electrophoresis (2D- proteins such as myc, src, fos, jun, myb, fms, PAGE): advances and perspectives. and raf-1. This is being carried out as an aid to Biotechniques. 2008 Apr;44(5):697-8, screening for the presence of these cancers. Further development underway is the 9. Ahmed FE Utility of mass spectrometry construction of proteomic maps of malignant for proteome analysis: part I. Conceptual tumor tissues to be used for diagnostic and experimental approaches. Expert Rev purposes, and to aid an early diagnosis and Proteomics. 2008 Dec;5(6):841-64. treatment optimization. 10. Seike M, Kondo T, Fujii K, et al. Additionally, several studies utilizing Proteomic signature of human cancer proteomics modalities and techniques is now cells. Proteomics 2004;4:2776-88. targeting many common disease processes 11. Tyers M & Mann M. From genomics to such as the diagnosis of chronic hepatitis, proteomics Nature, 2003, vol 422,13 whereby, scientific research have continued to reveal the protein biomarkers of serum 12. Platzer M. The human genome and its Hepatitis B and Hepatitis C viruses to aid in upcoming dynamics. Genome Dyn. the diagnosis and the control of therapeutic 13. Elias DR, Thorek DL, Chen AK, Validation of these new tests in large clinical Czupryna J, Tsourkas A. In vivo imaging trials is necessary prior to implementing of cancer biomarkers using activatable proteomics techniques and patterns routinely in molecular probes. Cancer Biomark. clinical use as tools for early disease detection. 2008;4(6):287-305. It is anticipated that databases of several 14. Liu YT. A technological update of proteins from tissues, cells and body fluids in molecular diagnostics for infectious 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 Raviglione MC.Consensus statement. and disease states that can be used for accurate Global burden of tuberculosis: estimated and rapid diagnosis39.
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Lichen planus

clinical practice Laurence Le Cleach, M.D., and Olivier Chosidow, M.D., Ph.D. This Journal feature begins with a case vignette highlighting a common clinical problem. Evidence supporting various strategies is then presented, followed by a review of formal guidelines, when they exist. The article ends with the authors' clinical recommendations.