C18 oasis hlb extraction cartridges

Protein extracts (100 μg) were concentrated on 30 K Filter-Aided Sample Preparation (FASP) filters (Expedeon). After washing with denaturing buffer (8 M urea in 100 mM Tris-HCl pH 8.5) at 10,000 rpm for 15 min, free thiol groups were alkylated by incubation with 50 mM iodoacetamide 30 min at room temperature in the dark. Then the filters were washed with denaturing buffer followed by washing with trypsin digestion buffer (50 mM ammonium bicarbonate pH 8.8). Protein samples were digested overnight at 37°C with sequencing grade trypsin (Promega, Madison, WI, USA) at 1:40 (w/w) trypsin: protein ratio in digestion buffer. The resulting tryptic peptides from each sample were recovered by centrifugation at 10,000 rpm for 5 min after the addition of 40 μl of trypsin digestion buffer, after which 50 μl of 500 mM NaCl was added and the filters centrifuged for 15 min at 10,000 rpm. Trifluoroacetic acid was added to a final concentration of 1%, and the peptides were desalted on C18 Oasis HLB extraction cartridges (Waters Corporation, Milford, MA, USA) and dried-down. Peptide labeling was performed using a 10-plex TMT kit (Tandem Mass Tag, Thermo Scientific, USA) according to the manufacturer's instructions, and desalted onto Oasis C18 cartridges before analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Peptide samples from DCs and EVs were labeled with 8-plex and 4-plex isobaric tags for relative quantitation (iTRAQ, Sigma-Aldrich), respectively, according to the manufacturer’s instructions. The individually labeled samples were mixed appropriately, desalted using C18 Oasis HLB extraction cartridges (Waters) and dried-down. Peptides from DCs were taken up in 0.1% TFA and separated into five fractions by high pH fractionation (25 (link)). The labeled peptide samples from EVs were taken up in 5 mM ammonium formate pH 3 with 15% acetonitrile (AcN, Sigma-Aldrich) and separated into six fractions by mixed-mode, strong cation-exchange, reversed-phase (26 (link)) fractionation on Oasis MCX cartridges (Waters).

The iTRAQ-labeled peptide samples obtained from DCs were taken up in immunoaffinity purification buffer (IPA buffer: 50 mM MOPS/NaOH pH 7.2, 10 mM Na₂HPO₄, 50 mM NaCl), sonicated and incubated with anti-K-Ac kit (PTMScan, Pilot Acetyl-Lysine Motif [Ac-K] Kit, CST #14499). Flow-through and eluted samples (the latter enriched in acetylated peptides) were desalted using C18 Oasis HLB extraction cartridges (Waters) and ZipTip pipette tips (Millipore), respectively, and dried-down.