Objective  To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS).

  Methods  The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The μLPickUp sample loading mode was used and only 1 μg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. (V/V). Acclaim^® PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. (V/V) with flow rate of 0.3 μL/min. Positive APCI nanospray interface was used and “one-drive-ten” schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed.

  Results  In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 μg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed.

  Conclusion  The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.