Morphology and proteomic analysis of leukocyte-free apheresis-derived exosome in storage

Objective To investigate the morphological and proteomic differences in exosomes (EXOs) during the storage of leukocyte-free apheresis platelets (LFA-Plt), evaluate the quality of platelets in storage and predict the function of EXOs at different storage periods. Methods EXOs were isolated by ultracentrifugation, then the morphological observation was performed by electron microscope. Particle size analysis and WB protein index detection were performed. 4D Label-free quantitative proteomics technology was used to perform quantitative and bioinformatics analysis on identified proteins. Protein differential analysis on the LFA- Plt EXO between group day 3 and day 5 was performed, and GO function and KEGG pathway enrichment analysis on differential proteins was conducted. Results Cup shaped, CD9/TSG101 enriched and Calnexin (-) EXO was successfully obtained. The particle size (nm) of LFA-Plt EXO for day 3 and day 5 were (82.2±19.6) and (83.4±19.4) respectively, and the protein concentration(μ g/uL) were (0.55±0.13) and (0.51±0.08) respectively, with no statistically significant difference between two groups (P>0.05). 1 504 proteins were identified in all samples. GO functional enrichment analysis showed that the LFA-Plt EXO proteins were mainly concentrated in the cell membrane, extracellular domain and proteasome core complex, and were related to the binding ability of proteins, ATP, calcium ions and GTP, and mainly participated in processes such as redox, protein hydrolysis and signal transduction. KEGG functional annotation showed that the EXO proteins mainly participated in material transportation and catabolism, genetic information processing, and were closely related to human tumors and viral bacterial infections, affecting the metabolism of human immune system. Compared with day 3, day 5 EXO showed significant up-regulation in 16 proteins. The GO enrichment analysis showed that 16 upregulated proteins were mainly associated with adenosine homocysteine activity and 6-phosphofructose kinase activity, and were mainly involved in the metabolism of organic nitrogen compound. KEGG enrichment pathway analysis showed that the most important function of upregulated proteins was participating in signaling pathway for oocyte maturation mediated by progesterone. Conclusion Under the preparation and storage conditions of LFA-Plt in our center, platelet quality can be relatively stable. The functions of EXO proteins varies with different storage periods, which may affect the effectiveness of platelet transfusion.