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PubMed2002

Identification of end points relevant to detection of potentially adverse drug reactions.

Expectations are high that the use of proteomics, gene arrays and metabonomics will improve risk assessment and enable prediction of toxicity early in drug development. These molecular profiling techniques may be used to classify compounds and to identify predictive markers that can be used to screen large numbers of chemicals. One of the challenges for the scientific community is to discriminate between changes in gene/protein expression and metabolic profiles reflecting physiological/adaptive responses, and changes related to pathology and toxicology. In these proceedings we provide a brief overview of the technologies with focus on proteomics and the possible applications to mechanistic and predictive toxicology. The discussion also includes strengths and limitations of molecular profiling technologies.

Animals↗
PubMed2002

Biomarkers, yesterday, today and tomorrow: the basis for health claims.

The development of useful and accurate biomarkers for predicting outcomes of food based interventions is becoming more and more important, given the emphasis being placed on ingredients in foods contributing to disease risk reduction and optimal health promotion. With the human genome now laid bare, opportunities abound to barcode individuals with their risk profiles. The massive increase in DNA sequence information together with the development of new technologies such as genomics, proteomics and bioinformatics, has resulted in a much greater capacity to determine individual risk profiles. Screening for biomarkers at the gene or protein expression level using microarray technology has the potential to identify new biomarkers for disease diagnosis. Whether these techniques will enable a better understanding of food-gene interactions to permit health claims rather than better therapeutic treatment (at high economic cost) remains to be demonstrated.

Biomarkers↗
PubMed2006

Analytical platforms for activity-based protein profiling--exploiting the versatility of chemistry for functional proteomics.

The field of proteomics aims to develop and apply technologies for the characterization of protein function on a global scale. Toward this end, synthetic chemistry has played a major role by providing new reagents to profile segments of the proteome based on activity rather than abundance. Small molecule probes for activity-based protein profiling have been created for more than a dozen enzyme classes and used to discover several enzyme activities elevated in disease states. These innovations have inspired complementary advancements in analytical chemistry, where new platforms have been introduced to augment the information content achievable in chemical proteomics experiments. Here, we will review these analytical platforms and discuss how they have exploited the versatility of chemical probes to gain unprecedented insights into the function of proteins in biological samples of high complexity.

Animals↗
PubMed2005

Mass spectrometry-based quantitative proteomic profiling.

Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis - such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner--is expected in the near future.

Biotin↗
PubMed2001

Solution and chip arrays in protein profiling.

The industrialization of proteomics demands reproducible, robust and high-throughput profiling technologies that current two-dimensional gel electrophoresis cannot achieve. New technologies in protein arrays, either on chips or with self-encoded elements in solution, hold much promise for interrogating the diverse and immense proteome.

Biosensing Techniques↗
PubMed2006

The burden of scientific progress: growing inequalities in the delivery of cancer care.

Novel targeted therapies are now a reality in oncology. The monoclonal antibodies trastuzumab, rituximab, cetuximab and bevacizumab have a proven benefit in both advanced and early stage cancers. Furthermore impressive new technologies such as gene profiling, circulating tumour cells and proteomics are becoming major tools for prognostic assessment and prediction of response to certain treatments. Whilst this is certainly good news for patients it comes at a price, many of these technologies are very expensive and may result in unprecedented inequalities in the delivery of cancer care. The largest impact may be on developing countries, but inequalities are starting to be felt in some developed nations. Strategies to tackle the resource implications need to be addressed alongside the development of these technologies.

Antibodies, Monoclonal↗
PubMed2003

Non-invasive methods of diagnosis of endometriosis.

PURPOSE OF REVIEW: Laparoscopy is the gold standard for the diagnosis of endometriosis but the need for visual evidence of the disease is a major stumbling-block for both effective clinical management of affected patients as well as for research into this common and debilitating reproductive disease. Laparoscopy is invasive and often causes a delay in diagnosis and treatment, especially in symptomatic teenagers and young women. Moreover, the visual inspection of the pelvis has major limitations, particularly for the diagnosis of retroperitoneal lesions. It is therefore not surprising that considerable efforts are being made to improve imaging techniques and to evaluate the diagnostic value of potential molecular markers of disease. RECENT FINDINGS: High-resolution transvaginal ultrasonography and, in selected cases, magnetic resonance imaging improve the diagnosis of retroperitoneal pelvic endometriosis as well as the identification of lesions that involve pelvic organs. A variety of serum and endometrial markers are being evaluated for their diagnostic potential, particularly in endometriosis associated infertility. The first gene profiling studies are showing positive results and proteomic technology is being applied to identify novel diagnostic protein expression patterns. SUMMARY: Current imaging techniques, such as transvaginal ultrasonography, are useful to screen the pelvis for the presence of retroperitoneal endometriosis but fail to diagnose peritoneal lesions, small ovarian endometriomas and adhesions. Postgenomic technologies and identification of novel serum and endometrial markers are likely to revolutionize future diagnosis of endometriosis.

Biomarkers↗
PubMed2005

Complex proteome prefractionation using microscale solution isoelectrofocusing.

The proteomes of mammalian cells, tissues and biologic fluids are complex and consist of proteins present over a wide dynamic range. Current protein profiling technologies do not have the capacity to overcome the sample complexity for comprehensive analysis of complex proteomes. A common strategy to substantially expand protein profiling capacities is sample prefractionation. A prefractionation method developed in the authors' laboratory, microscale solution isoelectrofocusing, has resulted in a commercial product, the ZOOM IEF Fractionator, which provides a simple and convenient method for high-resolution separation of complex proteomes based upon their isoelectric points. Complex human samples such as cancer cells and biologic fluids can be fractionated into well-resolved fractions with minimal cross-contamination of proteins between adjacent fractions. This review focuses on the application of microscale solution isoelectrofocusing prefractionation and subsequent downstream strategies in expanding protein profiling capacities and mining low-abundance proteins of complex proteomes.

Animals↗
PubMed2005

Modeling of protein signaling networks in clinical proteomics.

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomic endpoints in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states. One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor microenvironment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination, clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.

Humans↗
PubMed2003

Molecular diagnostics.

It is increasingly evident that molecular diagnostics, that is, the use of diagnostic testing to understand the molecular mechanisms of an individual patient's disease, will be pivotal in the delivery of safe and effective therapy for many diseases in the future. A huge body of new information on the genetic, genomic and proteomic profiles of different hematopoietic diseases is accumulating. This chapter focuses on new technologies and advancements in understanding the molecular basis of hematologic disorders, providing an overview of new information and its significance to patient care. In Section I, Dr. Braziel discusses the impact of new genetic information and research technologies on the actual practice of diagnostic molecular hematopathology. Recent and projected changes in methodologies and analytical strategies used by clinical molecular diagnostics laboratories for the evaluation of hematologic disorders will be discussed, and some of the challenges to clinical implementation of new molecular information and techniques will be highlighted. In Section II, Dr. Shipp provides an update on current scientific knowledge in the genomic profiling of malignant lymphomas, and describes some of the technical aspects of gene expression profiling. Analysis methods and the actual and potential clinical and therapeutic applications of information obtained from genomic profiling of malignant lymphomas are discussed. In Section III, Dr. Liotta presents an update on proteomic analysis, a new and very active area of research in hematopoietic malignancies. He describes new technologies for rapid identification of different important proteins and protein networks, and the potential therapeutic and prognostic value of the elucidation of these proteins and protein pathways in the clinical care of patients with malignant lymphomas.

Cytogenetic Analysis↗
PubMed2000

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

Animals↗
PubMed2006

Neuroblastoma detection using serum proteomic profiling: a novel mining technique for cancer?

BACKGROUND: Serum proteins in neuroblastoma (NB), such as neuron-specific enolase and lactate dehydrogenase, are used as nonspecific markers of disease severity. In this study, we have generated serum protein profiles that correlate with NB by applying proteomic technologies to uncover, at the protein level, serum polypeptide expression patterns in patients with NB. METHODS: Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry was used to generate protein expression spectra in human NB (group I, n = 18) and healthy children (group II, n = 17) sera. Groups I and II mass spectral data were compared after baseline subtraction. Peaks with high signal-to-noise ratios were selected and grouped into bins with various intervals along mass-to-charge axis. Two-sample t test and 3-fold cross validation were used to identify differential biomarkers between groups I and II. RESULTS: Significant differentially expressed proteins were identified between groups I and II (P < or = .05). The discriminatory features (proteomic patterns) of cancer from normal sera were successfully identified using the classification algorithm. The average classification performance after 3-fold cross validation was 87.26%. CONCLUSION: Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry is suitable for preliminary assessment of NB and could potentially provide a noninvasive diagnosis of NB. We propose that surface-enhanced laser desorption/ionization provides a novel method for NB diagnosis because direct observations of spectral differences between normal and NB sera are possible.

Child, Preschool↗
PubMed2005

Recent advances in the molecular diagnosis of diffuse large B-cell lymphoma.

The field of molecular diagnostics is changing and adapting to new information obtained from genetic, genomic and proteomic profiling of diseases. One of the novel technologies that has made significant impact on the molecular diagnosis of lymphoid malignancies is DNA microarray technology. It has allowed the profiling of the most common types of lymphomas, identifying distinct molecular signatures of these diseases as well as novel subtypes that cannot otherwise be identified by conventional methods. In addition, it has also allowed the construction of molecularly defined prognostic models for various types of lymphomas and to better understand the molecular mechanisms that determine the behavior of the tumor. In this review, recent advances in the molecular diagnosis of diffuse large B-cell lymphoma are highlighted, using examples of how gene expression profiling has been used in disease classification and outcome predictions. The future development of this field and its applications in the clinical arena will also be discussed.

Animals↗
PubMed2005

Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.

BACKGROUND & AIMS: Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. METHODS: To systematically study protein expression in pancreatic cancer, we used isotope-coded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. RESULTS: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. CONCLUSIONS: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.

Humans↗
PubMed2006

Analysis of the effects of cryopreservation on rat hepatocytes using SELDI-TOF mass spectrometry.

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Animals↗
PubMed2005

Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics.

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.

Animals↗
PubMed2005

Microarray-based functional protein profiling using peptide nucleic acid-encoded libraries.

The availability of complete genome sequences from numerous organisms has provided investigators with the challenge of assigning physiologic functions to the encoded gene products. To facilitate this process, multiple technologies have been developed to profile the transcriptome and the proteome, including methods to monitor the function of enzymes in complex biologic systems. These methods typically target specific classes of enzymes and attempt to correlate the enzymatic activity with the specific phenotype of interest. Here, technologies to measure enzymatic activity on a subproteomic scale are reviewed, including the authors' own efforts, which are based on self-assembled microarrays utilizing peptide nucleic acid-encoded small-molecule libraries.

Animals↗
PubMed2005

Quality control and quality assessment of data from surface-enhanced laser desorption/ionization (SELDI) time-of flight (TOF) mass spectrometry (MS).

BACKGROUND: Proteomic profiling of complex biological mixtures by the ProteinChip technology of surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) is one of the most promising approaches in toxicological, biological, and clinic research. The reliable identification of protein expression patterns and associated protein biomarkers that differentiate disease from health or that distinguish different stages of a disease depends on developing methods for assessing the quality of SELDI-TOF mass spectra. The use of SELDI data for biomarker identification requires application of rigorous procedures to detect and discard low quality spectra prior to data analysis. RESULTS: The systematic variability from plates, chips, and spot positions in SELDI experiments was evaluated using biological and technical replicates. Systematic biases on plates, chips, and spots were not found. The reproducibility of SELDI experiments was demonstrated by examining the resulting low coefficient of variances of five peaks presented in all 144 spectra from quality control samples that were loaded randomly on different spots in the chips of six bioprocessor plates. We developed a method to detect and discard low quality spectra prior to proteomic profiling data analysis, which uses a correlation matrix to measure the similarities among SELDI mass spectra obtained from similar biological samples. Application of the correlation matrix to our SELDI data for liver cancer and liver toxicity study and myeloma-associated lytic bone disease study confirmed this approach as an efficient and reliable method for detecting low quality spectra. CONCLUSION: This report provides evidence that systematic variability between plates, chips, and spots on which the samples were assayed using SELDI based proteomic procedures did not exist. The reproducibility of experiments in our studies was demonstrated to be acceptable and the profiling data for subsequent data analysis are reliable. Correlation matrix was developed as a quality control tool to detect and discard low quality spectra prior to data analysis. It proved to be a reliable method to measure the similarities among SELDI mass spectra and can be used for quality control to decrease noise in proteomic profiling data prior to data analysis.

Female↗