Abstract
Insulin secretory granules (ISGs) are cytoplasmic organelles of pancreatic beta-cells. They are responsible for the storage and secretion of insulin. To date, only about 30 different proteins have been clearly described to be associated with these organelles. However, data from two-dimensional gel electrophoresis analyses suggested that almost 150 different polypeptides might be present within ISGs. The elucidation of the identity and function of the ISG proteins by proteomics strategies would be of considerable help to further understand some of the underlying mechanisms implicated in ISG biogenesis and trafficking. Furthermore it should give the bases to the comprehension of impaired insulin secretion observed during diabetes. A proteomics analysis of an enriched insulin granule fraction from the rat insulin-secreting cell line INS-1E was performed. The efficacy of the fractionation procedure was assessed by Western blot and electron microscopy. Proteins of the ISG fraction were separated by SDS-PAGE, excised from consecutive gel slices, and tryptically digested. Peptides were analyzed by nano-LC-ESI-MS/MS. This strategy identified 130 different proteins that were classified into four structural groups including intravesicular proteins, membrane proteins, novel proteins, and other proteins. Confocal microscopy analysis demonstrated the association of Rab37 and VAMP8 with ISGs in INS-1E cells. In conclusion, the present study identified 130 proteins from which 110 are new proteins associated with ISGs. The elucidation of their role will further help in the understanding of the mechanisms governing impaired insulin secretion during diabetes.
Highlights
Insulin secretory granules (ISGs) are cytoplasmic organelles of pancreatic -cells
Western blot analyses of the obtained ISG fractions revealed the presence of calreticulin, indicating an endoplasmic reticulum (ER) contamination
To determine the enriched ISG fraction in the Nycodenz gradient, insulin was quantified in 12 fractions
Summary
Materials—All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich and were of the highest purity available. The Nycodenz enriched insulin granule fraction was recovered and loaded on a 27% Percoll solution, which was centrifuged at 35,000 ϫ g for 45 min. Equal amounts of proteins from the total cells and from the different cellular fractions were solubilized in sample buffer (62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, 10% (v/v) glycerol, 0.1 M dithiothreitol, and traces of bromphenol blue) before separation on a 12.5% T, 2.6% C polyacrylamide gel [71]. Cells were washed three times with PBS, blocked for 15 min in 10% FCS, and exposed for 45 min in PBS containing primary antibodies, 0.1% saponin, and 10% FCS. Cells were exposed to fluorescent dye-conjugated secondary antibodies (1:100) for 30 min at room temperature.
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