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Acinar cystic transformation (ACT) of the pancreas, previously called acinar cell cystadenoma, is a poorly understood and rare entity among pancreatic cystic lesions. This study aims to clarify its real nature. This research cohort included 25 patients with pancreatic ACT, representing the largest series in the literature. We describe their clinicopathological features and molecular profile using next-generation sequencing. ACT arose more often in women (F/M≃2:1), in the body-tail region, with a mean size of ~4 cm. At the latest follow-up, all patients were alive and disease free. Histologically, a typical acinar epithelium lined all cysts, intermingled with ductal-like epithelium in 11/25 (44%) cases. All the cases lacked any evidence of malignancy. Three ACT showed peculiar features: 1 showed an extensive and diffuse microcystic pattern, and the other 2 harbored foci of low-grade pancreatic intraepithelial neoplasia (PanIN) in the ductal-like epithelium. Next-generation sequencing revealed the presence of 2 pathogenic/likely pathogenic mutations in 2 different cases, 1 with ductal-like epithelium and 1 with PanIN, and affecting KRAS (c.34G>C, p.G12R) and SMO (c.1685G>A, p.R562Q) genes, respectively. The other case with PanIN was not available for sequencing. Overall, our findings support that ACT is a benign entity, potentially arising from heterogeneous conditions/background, including: (1) acinar microcysts, (2) malformations, (3) obstructive/inflammatory setting, (4) genetic predisposition, (5) possible neoplastic origin. Although all indications are that ACT is benign, the potential occurrence of driver mutations suggests discussing a potential role of long-term surveillance for these patients.

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Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, whose molecular pathogenesis has still not been well established. For the first time, a proteomic approach was undertaken to analyse the protein profiles of SMZL tissue. 1D and 2D Western blot, immunohistochemical analysis, and functional data mining were also performed in order to validate results, investigate protein species specific regulation, classify proteins, and explore their potential relationships. We demonstrated that SMZL is characterized by modulation of protein species related to energetic metabolism and apoptosis pathways. We also reported specific protein species (such as biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, and Set nuclear oncogene) directly involved in NF-kB and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Our findings shed new light on SMZL pathogenesis and provide a basis for the future development of novel biomarkers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD001124.

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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.

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In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations.

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To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes.

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Macrophages play a critical role at the crossroad between iron metabolism and immunity, being able to store and recycle iron derived from the phagocytosis of senescent erythrocytes. The way by which macrophages manage non-heme iron at physiological concentration is still not fully understood. We investigated protein changes in mouse bone marrow macrophages incubated with ferric ammonium citrate (FAC 10 µM iron). Differentially expressed spots were identified by nano RP-HPLC-ESI-MS/MS. Transcriptomic, metabolomics and western immunoblotting analyses complemented the proteomic approach. Pattern analysis was also used for identifying networks of proteins involved in iron homeostasis. FAC treatment resulted in higher abundance of several proteins including ferritins, cytoskeleton related proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the membrane level, vimentin, arginase, galectin-3 and macrophage migration inhibitory factor (MIF). Interestingly, GAPDH has been recently proposed to act as an alternative transferrin receptor for iron acquisition through internalization of the GAPDH-transferrin complex into the early endosomes. FAC treatment also induced the up-regulation of oxidative stress-related proteins (PRDX), which was further confirmed at the metabolic level (increase in GSSG, 8-isoprostane and pentose phosphate pathway intermediates) through mass spectrometry-based targeted metabolomics approaches. This study represents an example of the potential usefulness of "integarated omics" in the field of iron biology, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions. This article is part of a Special Issue entitled: Integrated omics.

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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.

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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.

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Arabidopsis halleri has the rare ability to colonize heavy metal-polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant-microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal-contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere-derived microorganisms. The results of this analysis emphasized the role of plant-microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2-DE and MS, indicated a general upregulation of photosynthesis-related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy-demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross-talk between heavy metal signaling and defense signaling.

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Mapping the proteome of microrganisms by 2D-electrophoresis is often a hard task, because many contaminants, e.g. polysaccharides of the cell wall and nucleic acid, can obstruct the pores of the IEF gel resulting in streaks and smears. A protocol based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) and its salt-dependent solubility was developed. The cellulose-producing strain Gluconoacetobacter hansenii AAB0248 was resolved on 7cm Minigels in over 500 protein spots (a hundred more than with protocols reported in literature). The method was further employed for mapping the proteome of some acid adapted, wine spoilage microrganisms e.g. acetic acid bacteria and a yeast.

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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 x 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.