Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research

The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.     

Current technologies to identify protein kinase substrates in high throughput

Since the discovery of protein phosphorylation as an important modulator of many cellular processes, the involvement of protein kinases in diseases, such as cancer, diabetes, cardiovascular diseases, and central nervous system pathologies, has been extensively documented. Our understanding of many disease pathologies at the molecular level, therefore, requires the comprehensive identification of substrates targeted by protein kinases. In this review, we focus on recent techniques for kinase substrate identification in high throughput, in particular on genetic and proteomic approaches. Each method with its inherent advantages and limitations is discussed.     

Screening and synthesis: high throughput technologies applied to parasitology

High throughput technologies continue to develop in response to the challenges set by the genome projects. This article discusses how the techniques of both high throughput screening (HTS) and synthesis can influence research in parasitology. Examples of the use of targeted and phenotype-based HTS using unbiased compound collections are provided. The important issue of identifying the protein target(s) of bioactive compounds is discussed from the synthetic chemist's perspective. This article concludes by reviewing recent examples of successful target identification studies in parasitology.     

South Africa: from species cradle to genomic applications

The South African government is committed to science and technology innovation, to establishing a knowledge-based economy and to harnessing life-sciences research for health and economic development. Given the constraints and the early stage of development of the field as a whole in South Africa, we found an impressive amount of research on human genomic variation in this country. Encouragingly, South Africa is beginning to apply genomics to address local health needs, including HIV and tuberculosis (TB) infections. We document a number of initiatives in South Africa that are beginning to study genetic variation within the various local indigenous populations. Other early initiatives focus on pharmacogenetic studies, mutation characterization in individual disease genes and genome-wide association studies. Public engagement in genomic issues is spear-headed by The Africa Genome Education Institute.     

Multiplex proteomic approaches to sepsis research: case studies employing new technologies

Sepsis is a multifactorial disease that provides unique challenges to the critical care physician. Diagnosis is hampered by the lack of a quantitative in vitro diagnostic test, instead, it relies on a series of clinical measures. The complex nature of the disease, with involvement of several physiologic systems, suggests a need to simultaneously monitor many clinical parameters. Novel proteomic technologies now exist that enable the multiplex measurement of multiple protein analytes from the same sample. Integration of these analytical measures with patient clinical data may provide the foundation for a better understanding of disease diagnosis, disease progression and the selection of optimal therapeutic regimen. The future challenge is the translation of these multiplex approaches from investigative research to clinical diagnostics for the greatest impact on patient treatment decisions.     

Multiplex proteomic approaches to sepsis research: case studies employing new technologies

Sepsis is a multifactorial disease that provides unique challenges to the critical care physician. Diagnosis is hampered by the lack of a quantitative in vitro diagnostic test, instead, it relies on a series of clinical measures. The complex nature of the disease, with involvement of several physiologic systems, suggests a need to simultaneously monitor many clinical parameters. Novel proteomic technologies now exist that enable the multiplex measurement of multiple protein analytes from the same sample. Integration of these analytical measures with patient clinical data may provide the foundation for a better understanding of disease diagnosis, disease progression and the selection of optimal therapeutic regimen. The future challenge is the translation of these multiplex approaches from investigative research to clinical diagnostics for the greatest impact on patient treatment decisions.     

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in –omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 μg dry TMT per channel was used to label 6–12 μg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.     

Integrating process engineering and microbiology tools to advance activated sludge wastewater treatment research and development

Wastewater treatment is a huge industryworldwide. Despite the massive capital andoperating costs, only a relatively small amountof R&D investment is made. This might havebeen related to the limited demands in terms ofeffluent quality in the past, but today'senvironmental awareness requires much strictereffluent standards to be achieved. This inturn should give sufficient incentives,together with the possible large cost savings,to increase the R&D activities in this field. There are certainly significant knowledge gapsto be filled and substantial benefits could begained from this.A range of knowledge gaps are identified inthis paper, extending from the role ofintermediates in nutrient removal overparameter estimation in modelling andsimulation to understanding the microbialmetabolic pathways at a genetic and enzymaticlevel. These gaps are opportunities andchallenges for all researchers andprofessionals in this field. Addressing themwill help substantially in the continuingdevelopment of wastewater treatmenttechnologies.The complexity of biological wastewatertreatment processes requires a broad range oftools and expertise to address the knowledgegaps. Novel process analysis tools arecritically important to investigate biologicaltreatment processes in future. They will comefrom different expertise areas and will need tobe used in close integration to gain maximalbenefits from the efforts. These tools willlikely include respirometry, novel chemicalanalyses, microsensors, gene-basedidentification, microbial physiology techniquesand integrated modelling and simulation. Examples of the application of such techniquesare provided to demonstrate the way thesetechniques may be used in future. In the nextfew years, there is likely an exciting andhighly interactive period of research anddevelopment for the wastewater industry.     

SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications

Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry.     

Comparison of high throughput screening technologies for luminescence cell-based reporter screens

As higher density formats become more and more common in HTS labs, the expectations for maintaining faster, lower cost screens puts great pressure on traditional 96-well screens. In some cases higher density formats are not compatible with the assay. This seems especially true in cell-based assays. In our case, the nature of the cells' response forced us to remain in 96-well plates. In this paper, we describe the development of a luminescence reporter assay and its performance in two detection modes, flash and glow. The advantages in cost and throughput for each technique are explored, along with automation considerations. An additional new technology, the use of pins for low-volume transfers, is also briefly described because of its dramatic effect on our screen's throughput. However, it will be more thoroughly presented in a future publication. Comparing the technologies available for HTS aids in designing automated systems that meet the unique needs of each assay.     

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.     

Monolithic capillary columns for liquid chromatography-electrospray ionization mass spectrometry in proteomic and genomic research

Peptides, proteins, single-stranded oligonucleotides, and double-stranded DNA fragments were separated with high resolution in micropellicular, monolithic capillary columns prepared by in situ radical copolymerization of styrene and divinylbenzene. Miniaturized chromatography both in the reversed-phase and the ion-pair reversed-phase mode could be realized in the same capillary column because of the nonpolar character of the poly-(styrene/divinylbenzene) stationary phase. The high chromatographic performance of the monolithic stationary phase facilitated the generation of peak capacities for the biopolymers in the range of 50-140 within 10 min under gradient elution conditions. Employing volatile mobile phase components, separations in the two chromatographic separation modes were on-line hyphenated to electrospray ionization (tandem) mass spectrometry, which yielded intact accurate molecular masses as well as sequence information derived from collision-induced fragmentation. The inaccuracy of mass determination in a quadrupole ion trap mass spectrometer was in the range of 0.01-0.02% for proteins up to a molecular mass of 20000, and 0.02-0.12% for DNA fragments up to a molecular mass of 310000. High-performance liquid chromatography-electrospray ionization mass spectrometry utilizing monolithic capillary columns was applied to the identification of proteins by peptide mass fingerprinting, tandem mass spectrometric sequencing, or intact molecular mass determination, as well as to the accurate sizing of double-stranded DNA fragments ranging in size from 50 to 500 base pairs, and to the detection of sequence variations in DNA fragments amplified by the polymerase chain reaction.     

An efficient,simple and high throughput protocol for cotton genomic DNA isolation

Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection.     

Recent advances in proteomic technologies applied to cardiovascular disease

In recent years, the diagnosis of cardiovascular disease (CVD) has increased its potential, also thanks to mass spectrometry (MS) proteomics. Modern MS proteomics tools permit analyzing a variety of biological samples, ranging from single cells to tissues and body fluids, like plasma and urine. This approach enhances the search for informative biomarkers in biological samples from apparently healthy individuals or patients, thus allowing an earlier and more precise diagnosis and a deeper comprehension of pathogenesis, development and outcome of CVD to further reduce the enormous burden of this disease on public health. In fact, many differences in protein expression between CVD‐affected and healthy subjects have been detected, but only a few of them have been useful to establish clinical biomarkers because they did not pass the verification and validation tests. For a concrete clinical support of MS proteomics to CVD, it is, therefore, necessary to: ameliorate the resolution, sensitivity, specificity, throughput, precision, and accuracy of MS platform components; standardize procedures for sample collection, preparation, and analysis; lower the costs of the analyses; reduce the time of biomarker verification and validation. At the same time, it will be fundamental, for the future perspectives of proteomics in clinical trials, to define the normal protein maps and the global patterns of normal protein levels, as well as those specific for the different expressions of CVD. J. Cell. Biochem. 114: 7–20, 2012. © 2012 Wiley Periodicals, Inc.     

Integrating Community-Based Interventions to Reverse the Convergent TB/HIV Epidemics in Rural South Africa

The WHO recommends integrating interventions to address the devastating TB/HIV co-epidemics in South Africa, yet integration has been poorly implemented and TB/HIV control efforts need strengthening. Identifying infected individuals is particularly difficult in rural settings. We used mathematical modeling to predict the impact of community-based, integrated TB/HIV case finding and additional control strategies on South Africa’s TB/HIV epidemics. We developed a model incorporating TB and HIV transmission to evaluate the effectiveness of integrating TB and HIV interventions in rural South Africa over 10 years. We modeled the impact of a novel screening program that integrates case finding for TB and HIV in the community, comparing it to status quo and recommended TB/HIV control strategies, including GeneXpert, MDR-TB treatment decentralization, improved first-line TB treatment cure rate, isoniazid preventive therapy, and expanded ART. Combining recommended interventions averted 27% of expected TB cases (95% CI 18–40%) 18% HIV (95% CI 13–24%), 60% MDR-TB (95% CI 34–83%), 69% XDR-TB (95% CI 34–90%), and 16% TB/HIV deaths (95% CI 12–29). Supplementing these interventions with annual community-based TB/HIV case finding averted a further 17% of TB cases (44% total; 95% CI 31–56%), 5% HIV (23% total; 95% CI 17–29%), 8% MDR-TB (68% total; 95% CI 40–88%), 4% XDR-TB (73% total; 95% CI 38–91%), and 8% TB/HIV deaths (24% total; 95% CI 16–39%). In addition to increasing screening frequency, we found that improving TB symptom questionnaire sensitivity, second-line TB treatment delays, default before initiating TB treatment or ART, and second-line TB drug efficacy were significantly associated with even greater reductions in TB and HIV cases. TB/HIV epidemics in South Africa were most effectively curtailed by simultaneously implementing interventions that integrated community-based TB/HIV control strategies and targeted drug-resistant TB. Strengthening existing TB and HIV treatment programs is needed to further reduce disease incidence.     

New tools for the high throughput characterization of rat genomic DNA samples

Linkage studies and positional cloning projects for the identification of disease related genes require the genetic characterization of large numbers of genomic DNA samples. The application of spotting robots enables the production of high density filters representing several thousand DNA probes. To take full advantage of the potential of these filters we have established a new, hybridization based Interspersed Repetitive Sequence (IRS-)marker system for the rat genome. This marker panel was shown to be useful for rapid genotyping of many multigenic crosses as well as high throughput characterization of large insert genomic libraries.     

Issues for cross-cultural psychiatric research in South Africa

South Africa's heterogenous society offers many opportunities for cross-cultural psychiatric research, but researchers in the country are subject to a number of restraints. Apart from legally enforced segregation, there are strict censorship laws and restricted access to certain types of information. The issues surrounding categorization of cultures and factors affecting publishing research from South Africa have important implications for the type of work that is done. It is a central argument of this article that the issues affecting research in South Africa are relevant to other countries as well, and parallels between the local and international context are drawn. The South African experience suggests that analysis of the research enterprise itself is a useful part of the business of cross-cultural psychiatric research.I am grateful to Beverley Jo Dickman, Terry Dowdall, Ronith Elk, Alan Flisher, Don Foster, Jane Steere and Sally Swartz for their comments on this article. Emile Boonzaaier provided a useful critique on an earlier draft. The arguments presented in this article are, however, my responsibility alone.     

Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.