Exosomes are membranous vesicles released by cells in extracellular fluids: they have been found and analyzed in blood, urine, amniotic fluid, breast milk, seminal fluid, saliva and malignant effusions, besides conditioned media from different cell lines. Several recent papers show that exosome proteomes of different origin include both a common set of membrane and cytosolic proteins, and specific subsets of proteins, likely correlated to cell-type associated functions. This is particularly interesting in relation to their possible involvement in human diseases. The knowledge of exosome proteomics can help not only in understanding their biological roles but also in supplying new biomarkers to be searched for in patients' fluids. This review offers an overview of technical and analytical issues in exosome proteomics, and it highlights the significance of proteomic studies in terms of biological and clinical usefulness.

It has previously been reported that growth hormone secretagogues (GHS) may have a role in the regulation of bone metabolism in animals and humans. In this study we evaluated the effect of ghrelin, the endogenous ligand of GHS receptors, on the proliferation rate and on osteoblast activity in primary cultures of rat calvaria osteoblasts. In the same experiments, we compared the effects of ghrelin with those of hexarelin (HEXA) and EP-40737, two synthetic GHS with different characteristics. Both ghrelin and HEXA (10 11 -10 8 M) significantly stimulated osteoblast proliferation at low concentrations (10 10 M). Surprisingly, EP-40737 demonstrated an antiproliferative effect at 10 9 -10 8 M, whereas lower concentrations had no effect on cell proliferation. Ghrelin and HEXA significantly increased alkaline phosphatase (ALP) and osteocalcin (OC) production. At variance with these peptides, EP-40737 did not significantly stimulate ALP and OC. The mRNA for GHS-R1a receptors and the corresponding protein were detected in calvarial osteoblasts by RT-PCR and Western blot respectively, indicating that ghrelin and GHS may bind and activate this specific receptor. We conclude that endogenous ghrelin and synthetic GHS modulate proliferation and differentiation of rat osteoblasts, probably by acting on their specific receptor.

aRenal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties.Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE.Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC‐Q‐TOF‐MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed‐phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynxTM (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q‐TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis.

The thermotropic behavior of palmitoylsphingomyelin vesicles containing GM1 ganglioside and cholesterol has been investigated by high-sensitivity differential scanning calorimetry. The thermograms exhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvable into two components. The relative contribution of the minor component, undetectable in the absence of ganglioside, to the total enthalpy and its transition temperature (>40 degrees C) increase with the concentration of the glycolipid embedded in the vesicles. These data suggest the occurrence of lateral phase separation and that more ordered, higher melting GM1 ganglioside-enriched domains are present within the sphingomyelin bilayer. Studies on binary sphingomyelin/cholesterol mixtures confirmed the known tendency of the sterol to decrease the total enthalpy of sphingomyelin, forming cholesterol-enriched domains. The thermograms exhibited by ternary sphingomyelin/ganglioside/cholesterol mixtures in variable proportions (up to 20% molar GM1 or Chol) displayed, on increasing the content of either the sterol or the ganglioside, features addressable to sphingomyelin/cholesterol (peaks centered at temperature 40 degrees C), respectively. This trend was confirmed by deconvolution analysis, showing that the thermograms are resolvable into components addressable to GM1-enriched and to cholesterol-enriched domains. Taken all together, the results show that the architectural features of sphingomyelin bilayers are strongly dependent on the presence of GM1 ganglioside and cholesterol, whose presence is leading to the formation of separate, GM1-enriched and cholesterol-enriched distinct domains. Ganglioside-sphingomyelin and sphingomyelin-cholesterol, together with mutual ganglioside-ganglioside, interactions could contribute to maintain a network of bonds extending to proteins, forming specialized membrane domains, such as caveolae, or others, whose experimental clues are the glycolipid-enriched detergent-insoluble fractions that can be isolated from cell membranes.