Gel-based proteomics in disease research: Is it still valuable?

Gel electrophoresis had been the primary method in proteomics. In the early era of proteomics, gel electrophoresis was a dominant technique of sample preparation for mass spectrometry analysis. Particularly, two-dimensional electrophoresis provided high-resolution proteome separation, and was regarded as the standard methodology for the separation of wide-range proteomes. However, gel electrophoresis turned downwards due to the progress of other separations including liquid chromatography and ionization techniques, resulting gel-free proteomics finally becoming dominant players at present. There are numerous advantages in gel-free approach in aspects of current trends of disease research. Interestingly, gel-free approaches are still advanced, it seems that gel electrophoresis will not be disappeared. The unique features of gel electrophoresis can be complementary for gel-free and it is suitable for the new wave of top-down functional proteomics.     

What place for polyacrylamide in proteomics?

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to deliver high quality protein resolution and dynamic range for the proteomics researcher. To remain as the preferred method for protein separation and characterization, several key steps need to be implemented to ensure quality sample preparation and speed of analysis. Here, we describe the progress made towards establishing 2D-PAGE as the optimal separation tool for proteomics research.     

Is proteomics heading in the wrong direction?

Proteomics is now considered to be one of the most important 'post-genome' approaches to help us understand gene function. In fact, several genomics companies have launched large-scale proteomics projects, and have started to annotate the entire human proteome. The 'holistic view' painted by a human proteome project is seductive, but is it realistic?     

When should one subtract background fluorescence in 2-color microarrays?

Two-color microarrays are a powerful tool for genomic analysis, but have noise components that make inferences regarding gene expression inefficient and potentially misleading. Background fluorescence, whether attributable to nonspecific binding or other sources, is an important component of noise. The decision to subtract fluorescence surrounding spots of hybridization from spot fluorescence has been controversial, with no clear criteria for determining circumstances that may favor, or disfavor, background subtraction. While it is generally accepted that subtracting background reduces bias but increases variance in the estimates of the ratios of interest, no formal analysis of the bias-variance trade off of background subtraction has been undertaken. In this paper, we use simulation to systematically examine the bias-variance trade off under a variety of possible experimental conditions. Our simulation is based on data obtained from 2 self versus self microarray experiments and is free of distributional assumptions. Our results identify factors that are important for determining whether to background subtract, including the correlation of foreground to background intensity ratios. Using these results, we develop recommendations for diagnostic visualizations that can help decisions about background subtraction.     

Why nestedness in mutualistic networks?

We investigate the relationship between the nested organization of mutualistic systems and their robustness against the extinction of species. We establish that a nested pattern of contacts is the best possible one as far as robustness is concerned, but only when the least linked species have the greater probability of becoming extinct. We introduce a coefficient that provides a quantitative measure of the robustness of a mutualistic system.     

DNA microarrays in the clinic: how soon,how extensively?

Although DNA microarrays are now widely used in research settings, they have been slow to penetrate clinical practice in spite of their apparent advantages. This is due to the very different requirements for a clinical test in contrast to a research tool, and to a strict necessity for demonstrated clinical utility. There is a clear differentiation between two types of DNA array tests: "genomic" diagnostics, developed to ascertain the presence or absence of mutations, deletions or duplications, and for which clinical evidence is already established, and tests using expression profiling for prognosis or predictive purposes, in which case the clinical correlate must be proven. Most array diagnostics currently used belong, understandably, to the "genomic" variety. It is to be expected that future improvements in tailored technology, as well as a logical trend towards measuring an ever-increasing number of parameters, will ensure an important diagnostic role for DNA arrays in the coming decade.     

What does it mean to identify a protein in proteomics?

The annotation of the human genome indicates the surprisingly low number of approximately 40,000 genes. However, the estimated number of proteins encoded by these genes is two to three orders of magnitude higher. The ability to unambiguously identify the proteins is a prerequisite for their functional investigation. As proteins derived from the same gene can be largely identical, and might differ only in small but functionally relevant details, protein identification tools must not only identify a large number of proteins but also be able to differentiate between close relatives. This information can be generated by mass spectrometry, an approach that identifies proteins by partial analysis of their digestion-derived peptides. Information gleaned from databases fills in the missing sequence information. Because both sequence databases and experimental data are limited, a certain ambiguity often remains concerning which sequence variant(s) and modification(s) are present. As the common denominator of all the isoforms is a gene, in our opinion, it would be more accurate to state that a product of this particular gene rather than a certain protein has been identified by mass spectrometry.     

Is proteomics the new genomics?

Mass spectrometry (MS)-based proteomics has become a formidable tool for the investigation of posttranslational modifications to proteins, protein interactions, and organelles. Is it now ready to tackle comprehensive protein expression analysis?     

Diagonal reverse-phase chromatography applications in peptide-centric proteomics: ahead of catalogue-omics?

Diagonal electrophoresis/chromatography was described 40 years ago and was used to isolate specific sets of peptides from simple peptide mixtures such as protease digests of purified proteins. Recently, we have adapted the core technology of diagonal chromatography so that the technique can be used in so-called gel-free, peptide-centric proteome studies. Here we review the different procedures we have developed over the past few years, sorting of methionyl, cysteinyl, amino terminal, and phosphorylated peptides. We illustrate the power of the technique, termed COFRADIC (combined fractional diagonal chromatography), in the case of a peptide-centric analysis of a sputum sol phase sample of a patient suffering from chronic obstructive pulmonary disease (COPD). We were able to identify an unexpectedly high number of intracellular proteins next to known biomarkers.     

Why does liver fibrosis occur in schistosomiasis?

Schistosomiasis is one of the most prevalent of several chronic inflammatory diseases in which morbidity results primarily from tissue scarring. New concepts regarding the molecular pathogenesis of scar formation are being applied in research efforts to define the basis of liver fibrosis in schistosomiasis. Such investigations have led to the identification of an apparently novel lymphokine, fibroblast stimulating factor-I (FsF-I), produced in the egg granulomas. FsF-1 and other granulomo-derived fibrogenic cytokines may represent the molecular links between periovular granulomotous inflammation and hepatic fibrosis. Here, David Wyler postulates that the unmodified production of these f brogenic signals may be responsible for the development of severe hepatic fibrosis in the subpopulotion of infected individuals who develop this complication.     

Why Don't Chimpanzees in Gabon Crack Nuts?

Some populations of wild chimpanzees (Pan troglodytes) use hammers and anvils of stone or wood to crack open nuts for food. Others do not. The aim of this study was to ask why one non-nut-cracking population, in the Lopé Reserve, Gabon, lacks this useful form of tool use. We tested 10 hypotheses: (1) nuts are absent; (2) nuts are few; (3) nuts are unsuitable; (4) hammers are absent; (5) hammers are unsuitable; (6) anvils are absent; (7) anvils are unsuitable; (8) nuts are displaced by better food items; (9) intelligence is insufficient; and (10) knowledge is insufficient. All but the last are clearly falsified, leaving by exclusion the likelihood that Lopé's chimpanzees lack the technology—knowledge of appropriate technique—to exploit this resource. Thus, the behavioral differences across populations of these apes are cultural and not environmentally dictated. This explanation is congruent with the distribution of chimpanzee nut-cracking across Africa.