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In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 μm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 μm i.d. nano-LC packing column with 3 μm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7–170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.

Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.

Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30–150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years. In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.

BackgroundLow-intensity pulsed ultrasound (LIPUS) can induce mesenchymal stem cell (MSC) differentiation, although the mechanism of its potential effects on chondrogenic differentiation is unknown. Since autophagy is known to regulate the differentiation of MSCs, the aim of our study was to determine whether LIPUS induced chondrogenesis via autophagy regulation.MethodsMSCs were isolated from the rat bone marrow, cultured in either standard or chondrogenic medium, and stimulated with 3 MHz of LIPUS given in 20% on–off cycles, with or without prior addition of an autophagy inhibitor or agonist. Chondrogenesis was evaluated on the basis of aggrecan (AGG) organization and the amount of type II collagen (COL2) and the mRNA expression of AGG, COL2, and SRY-related high mobility group-box gene 9 (SOX9) genes.ResultsLIPUS promoted the chondrogenic differentiation of MSCs, as shown by the changes in the extracellular matrix (ECM) proteins and upregulation of chondrogenic genes, and these effects were respectively augmented and inhibited by the autophagy inhibitor and agonist.ConclusionsTaken together, these results indicate that LIPUS promotes MSC chondrogenesis by inhibiting autophagy.

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We evaluated the effect of low-intensity pulsed ultrasound (LIPUS) on MMP-13 and MAPKs expression in rabbit knee osteoarthritis (OA). For this purpose, 18 New Zealand white rabbits were randomly and equally divided into O + L, O - L, and SO groups. In O + L group, animals underwent right back leg ACLT operation and LIPUS radiation. In O - L group, animals underwent ACLT but no LIPUS treatment. In SO (control) group, animals underwent sham operation without LIPUS. After 6 weeks, we assessed the pathologic changes in the articular surface of femoral condyle and compared using Mankin scores. Also, expression of type-II collagen, MMP-13, ERK1/2, p38, and JNK was measured by Western blot. Compared with controls, Mankin scores were higher in O + L (P < 0.05)/O - L (P < 0.01) groups. Compared with O + L group, score was higher in O - L group (P < 0.05). Compared with controls, type-II collagen expression was less in O + L/O - L groups, with more significant decrease in O - L group (P < 0.05). Contrarily, expression of MMP-13, p-ERK1/2, and p-p38 was enhanced in O + L/O - L groups as compared with controls, with more significant increase in O - L group (P < 0.01). Compared with O + L group, expression was higher in O - L group (P < 0.05). We, therefore, concluded that LIPUS application promoted cartilage repair in OA through the downregulation of MMP-13, ERK1/2, and p38.

The heterogeneous populations of exosomes with distinct nanosize have impeded our understanding of their corresponding function as intercellular communication agents. Profiling signaling proteins packaged in each size-dependent subtype can disclose this heterogeneity of exosomes. Herein, new strategy was developed for deconstructing heterogeneity of distinct-size urine exosome subpopulations by profiling N-glycoproteomics and phosphoproteomics simultaneously. Two-dimension size exclusion liquid chromatography (SEC) was utilized to isolate large exosomes (L-Exo), medium exosomes (M-Exo), and small exosomes (S-Exo) from human urine samples. Then, hydrophilic carbonyl-functionalized magnetic zirconium-organic framework (CFMZOF) was developed as probe for capturing the two kinds of post-translational modification (PTM) peptides simultaneously. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with database search was used to characterize PTM protein contents. We identified 144 glycoproteins and 44 phosphoproteins from L-Exo, 156 glycoproteins, and 46 phosphoproteins from M-Exo and 134 glycoproteins and 10 phosphoproteins from S-Exo. The ratio of the proteins with simultaneous glycosylation and phosphorylation is 11%, 9%, and 3% in L-Exo, M-Exo, and S-Exo, respectively. Based on label-free quantification intensity results, both principal component analysis and Pearson’s correlation coefficients indicate that distinct-size exosome subpopulations exist significant differences in PTM protein contents. Analysis of high abundance PTM proteins in each exosome subset reveals that the preferentially packaged PTM proteins in L-Exo, M-Exo, and S-Exo are associated with immune response, biological metabolism, and molecule transport processes, respectively. Our PTM proteomics study based on size-dependent exosome subtypes opens a new avenue for deconstructing the heterogeneity of exosomes.