). This opens for the possibility that cells in a context-dependent manner secrete subpopulations of organelle-specific EVs, and suggest that the lytic granules may be an alternative compartment for EV release in cytotoxic cells.
In this study, we aimed to isolate a subpopulation of cytolytic EVs from NK cells that may be exploited for treatment of solid tumors as a more potent EV product compared to bulk EVs. To this end, we fractionated NK-EVs from the human NK-92 cell lineeither density-gradient ultracentrifugation or size-exclusion chromatography . Comparative quantitative label-free proteomic analysis revealed unique protein landscapes of specific EV fractions separated by DG-UC.
SEC-separated NK-92 EVs or DG-UC-separated EVs from primary NK cells were lysed with 0.1% ProteaseMax Surfactant, after which the proteins were reduced, alkylated and digested into peptides with trypsin. The resulting peptide mixture was purified by STAGE-TIP method using a C18 resin disk before the samples were analyzed by a nanoLC-MS/MS using nanoElute coupled to timsTOF fleX with 60 min separation gradient and 25 cm Aurora C18 column.
MS raw files were submitted to MaxQuant software version 1.6.17.0 or version 2.0.3.0 for protein identification and label-free quantification . Carbamidomethyl was set as a fixed modification and acetyl , carbamyl and oxidation were set as variable modifications. First search peptide tolerance of 20 ppm and main search error 4.5 ppm were used. Trypsin without proline restriction enzyme option was used, with two allowed miscleavages.
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