Gene Ontology GO enrichment analysis was performed on the identified glycoproteins from HeLa cell exosomes to examine the cellular components, molecular function, and biological processes in which they are involved. For molecular function, most of the glycoproteins played an important role in protein binding and some of them related to receptor activity. This result indicated that the identified glycoproteins mainly originate from the plasma membrane and are associated with cell adhesion and migration. The hydrophilic ionic liquid was utilized for the synthesis of glycopeptides enrichment material for the first time.
The nanoparticles showed high specificity, selectivity, sensitivity and recovery yield for glycopeptide enrichment with a standard glycoprotein compared with the commercial material. This work provides a new avenue for glycoproteome research and broadens the research possibilities for the study of exosomes. All other chemical agents were obtained from Shanghai Chemical Reagent.
The resulting nanoparticles were collected with a magnet and washed sequentially with ethanol and water three times. The composite was separated by a magnet and washed sequentially with water and isopropanol three times. HeLa exosomes were isolated in the supernatant, which was obtained by differential ultracentrifugation of cultured cells Bovine serum used for culturing these cells was subjected to ultracentrifugation to remove exosomes prior to use. The cell culture medium was collected after each passage for exosome isolation. The method of exosome isolation is shown in Fig.
The morphology of extracted exosomes was investigated by transmission electron microscopy TEM. The digests were desalted prior to glycopeptide enrichment. The enrichment process was shown in Fig. Mobile phase A was composed of 0. The spray voltage was operated at 2. The mass tolerances were 20 ppm for initial precursor ions and 0. Two missed cleavages in tryptic digests were allowed.
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