Characterization of membrane-shed microvesicles from cytokine-stimulated β-cells using proteomics strategies

Abstract

Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular bodies with the plasma membrane, respectively. They are thought to be involved in many significant biological processes such as cell to cell communication, rescue from apoptosis, and immunological responses. Here we report for the first time a quantitative study of proteins from β-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of β-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from β-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, α-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting serine-threonine kinase 1, and intercellular adhesion molecule 1.

Fig. 1.

A, ApopTag images of pancreatic β-cells. The cells were treated as indicated under “Experimental Procedures.” The red cells represent labeled apoptotic cells with DAPI counterstaining of the nuclei. The magnification is 40×. B, relative values of β-cell apoptosis upon different treatments using staurosporine as a positive control. The values are based on death cell counts from 10× magnification images (data not shown) after 24 h of stimulation.

Fig. 2.

Transmission electron microscopy images of isolated β-cell-derived control and IL-1β/TNFα-stimulated exosomes and MPs. The arrows indicate the two types of microvesicles. The vesicles were fixed in paraformaldehyde and contrasted with uranyl oxalate. The pictures were obtained with 65,000× magnification. The scale bar is 200 nm.

Fig. 3.

Flow diagram of the strategy used to target the membrane proteins from microparticles and exosomes from control and stimulated β-cells.

Fig. 4.

A, overlap of the identified phosphorylated proteins from exosomes and MPs. B, overlap of the identified N-linked sialylated glycoproteins from exosomes and MPs. C, overlap of the total proteins from exosomes and MPs.

Fig. 5.

Network analysis of quantitative proteomic data derived from exosomes (A) and MPs (B) from control and stimulated β-cells. Network analysis of cytokine-stimulated β-cell microparticles and exosomes proteome reveals an increase in the levels of TNFR1 (A) and ICAM-1 (B) involved in cell death and cell signaling. The red- and green-filled nodes indicate an up- and down-regulation in cytokine-stimulated β-cells microvesicles. The various shapes of nodes denote the different functions.

Fig. 6.

A, tandem MS/MS spectrum of the peptide SLESGSNFAPLK from tumor necrosis factor receptor superfamily member 1A, identified as significantly “up-regulated” in the exosomes fraction. The MS signals are shown in the inset. B, tandem MS/MS spectrum of the heavy phosphorylated peptide SLEpSGSNFAPLK from tumor necrosis factor α-induced protein 3 identified as significantly “up-regulated” in the exosomes fraction. The MS signals are shown in the inset. C, tandem MSMS spectrum of the sialylated glycopeptide GNETLSR from ICAM-1. The MS signals are shown in the inset.