Amicon ultrafiltration tube

The UC (ultracentrifugation) method was optimized according to the previously described approach (25 (link)). After thawing at 37°C, plasma samples were centrifugated at 3,000 ×g for 15 min to remove cell debris. Then, the supernatant was diluted by a seven-fold volume of phosphate-buffered saline (PBS), centrifuged at 13,000 ×g for 30 min, and processed through a 0.22 μm filter to remove large particles. The supernatant was then ultracentrifuged using a P50A72-986 rotor (CP100NX; Hitachi, Brea, CA, USA) at 100,000 ×g, 4°C, for 2 h to pellet the exosomes. Then, the pellet was re-suspended in PBS and centrifuged at 100,000 ×g 4°C for 2 h. After PBS washing, the exosomes pellet was re-suspended in 100 µl PBS.
The size exclusion method (SEC) was used to separate the exosomes. Briefly, after 0.8 μm filtration, 1 ml plasma sample was diluted with 1.5 times PBS and then purified by Exosupur exclusion column (Echobiotech, China) following the manufacturer’s instructions. The sample was then eluted with 0.1M PBS, and 2ml of the target fraction was collected. Finally, the exosome solution was concentrated (200 μL) through an Amicon^® ultrafiltration tube with a molecular weight cutoff of 100kDa (Merck, Germany).

All samples were treated according to a previously published method [26 (link)]. Urine samples were removed from −80 °C storage and thawed at 4 °C, then centrifuged at 3000× g for 20 min at 4 °C to remove cells and debris. The sample was subsequently passed through a 15-mL Amicon ultrafiltration tube (Ultra-3k, Merck Millipore Ltd., Co. Cork, Ireland) and the flow-through was saved. Acetone (pre-chilled at −20 °C) was added to precipitate the proteins at −20 °C for 4 h. The precipitation was dissolved in 1% SDS/8M urea, and the protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). For this assay, the maximum amount of SDS allowed in the protein sample without causing a noticeable interference is 5% [30 ]. The protein lysate was reduced in 10 mM of Dithiothreitol (DTT) at 37 °C, alkylated with 20 mM of iodoacetamide (IAM) at room temperature in darkness, and digested with trypsin at 37 °C. Digested peptides were desalted with a C18 spin column (Merck KGaA, Darmstadt, Germany). Peptide concentration was determined using a Peptide Assay, 500 Assays (Thermo Scientific, Waltham, MA, USA). The digested peptides were fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific, Waltham, MA, USA). Fractions were pooled at various intervals to 10 fractions.

Pepsin from porcine stomach mucosa, dialysis membrane (14 kDa MWCO), DPPH, and type I collagen standard solutions from calf skin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alcalase^® 2.4 L (a proteinase from Bacillus licheniformis) was donated by Novozymes (Mexico City, Mexico). Papain enzyme from Carica papaya (30,000 U/mg) and Amicon ultrafiltration tubes (3 kDa MWCO) were purchased from Merck Corporation (Burlington, MA, USA). The protein marker and bovine serum albumin standard (2 mg mL⁻¹) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Solvents for amino acid analysis were HPLC-grade (T.J. Baker, Chemicals, PA, USA). All other chemicals used in this investigation were of analytical grade and used as received.