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The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of \"isobaric\" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

Background Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels. Results Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Conclusion This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.

Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7 cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18–24 cm) on 2-PE and report some representative images for a qualitative assessment.

Macrophages, originating from the migration and differentiation of circulating monocytes into virtually all tissues, are extremely flexible and plastic cells that play vital homeostatic roles, but also contribute to the pathophysiology of many human diseases. For these reasons, they are intensively studied by different approaches, recently including proteomics. Macrophage cells can be taken from a range of different sources, including blood monocytes and macrophages from tissues. Macrophages can also be generated by in vitro culture from blood monocytes, and cell lines derived from this lineage can be used. Similarly, many different proteomic techniques can be used, ranging from classic approaches based on 2D gel electrophoresis to more recent high-throughput gel-free techniques essentially based on mass spectrometry. Here, we review the application of such techniques to the study of monocytes/macrophages, and summarize some results potentially relevant to two paradigmatic conditions - atherosclerosis and disorders of iron metabolism.