Sequencing grade modified trypsin v511a

Referred to the procedure of Shevchenko et al. [46 (link)], but skipped the steps of reduction and alkylation. An Ettan automated spot picker from 2DE was used to pick the interesting spots, which were then put into 0.5 mL microtubes (Axygen; extrapure). The gel pieces were destained with 50 μL of 10 mM ammonium bicarbonate/50% acetonitrile and incubated with vortexing for 15 min. After removing the supernatant, 100 μL of 100% acetonitrile was added for gel pieces drying. Subsequently, the liquid was eliminated and 25 μL of 25 mM ammonium bicarbonate was added for 5 min., followed by a wash with 25 μL of 100% acetonitrile for 15 min. After removing all remaining liquid, 100 μL neat acetonitrile was added with vortexing for 3 min., until the gel pieces become white and shrinking. The gel pieces then were dried in a SpeedVac, and reswelled in 20 ng/μL sequencing grade modified procine trypsin (Promega sequencing grade modified trypsin (V511A)) in 10 mM ammonium bicarbonate for 1 h. Subsequently, the gel pieces were covered with 10mM ammonium bicarbonate and incubated at 37 °C overnight. Peptides were extracted by 15 μL of 50% acetonitrile with 1% trifluoroacetic acid for 15 min. twice, and then stored at −20 °C prior to LC-MS/MS or MALDI-TOF MS analysis.

Exosomal proteins of ADSC-exo and ADSC-F-exo (n = 2 for each kind of sample) were purified using the T-PER tissue protein extraction reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were desalted using the Amicon^® Ultra-15 (Merck-Millipore, Burlington, MA, USA) and quantified using the BCA protein assay (23225, Thermo Fisher Scientific). For iTRAQ labeling, 25 µg of the protein samples were dried using SpeedVac and resuspended in the iTRAQ dissolution buffer, which included 0.5 M triethylammonium bicarbonate (TEAB; pH 8.5). Protein samples underwent reduction using the iTRAQ reduction buffer (tris-2-carboxyethyl phosphine, TCEP) at 60 °C for 30 min and were then alkylated in the dark using iodoacetamide at 37 °C for 30 min. After protein digestion using sequencing grade modified trypsin (V511A, Promega, Madison, WI, USA), samples were dried using SpeedVac. Next, the peptides were reconstituted in the iTRAQ dissolution buffer and labeled using iTRAQ labeling reagents, according to the manufacturer’s instructions (Applied Biosystems Inc., Foster City, CA, USA).