Easy spray c18 pepmap

Approximately 50 μg of EV proteins were reduced with 10 mmol/L dithiothreitol for 25 minutes, alkylated with 50 mmol/L indole-3-acetic acid for 30 minutes in the dark, and precipitated overnight with 80% acetone. The precipitated pellet was washed once with 80% acetone, resuspended in 6 M urea/2 M and digested using LysC and trypsin. Digested peptides were cleaned using stage tips, eluted with 70% acetonitrile and lyophilized. Samples were resuspended in 0.1% formic acid and separated with a Thermo Fisher Scientific RSLCnano Ultimate 3000 LC on a Thermo Fisher Scientific Easy-Spray C18 PepMap 75 μm × 50 cm C-18 2 μmol/L column. A 75-minute gradient of 2%–25% (30 minutes) acetonitrile with 0.1% formic acid was run at 300 nL/minute at 50°C. Eluted peptides were analyzed by a Thermo Fisher Scientific Q Exactive mass spectrometer utilizing a top 15 methodology in which the 15 most intense peptide precursor ions were subjected to fragmentation. The automatic gain control for MS1 was set to 3 × 10⁶ with a max injection time of 120 ms, the automatic gain control for MS2 ions was set to 1 × 105 with a max injection time of 150 ms, and the dynamic exclusion was set to 90 seconds.

Mouse colon epithelial cells were harvested using lysis buffer (50 mM HEPES pH 8.0, 4% SDS). Protein quantification was performed using BCA protein assay and 100 μg of protein was digested using LysC for 3 hours, followed by trypsin overnight. Following day, digested peptides where isolated using C-18 and PGC columns, then dried and washed using ethyl acetate. Three μg were then resuspended in 0.1% formic acid and separated with a Thermo Scientific RSLC nano Ultimate 3000 LC on a Thermo Scientific Easy-Spray C-18 PepMap 75 mm × 50 cm C-18 2 mm column. A 305 min gradient of 2–20% (180 min) 20%–28% (45 min) 28%–48% (20 min) acetonitrile with 0.1% formic acid was run at 300 nL/min at 50C. Eluted peptides were analyzed by Thermo Scientific Q Exactive or Q Exactive plus mass spectrometers utilizing a top 15 methodology in which the 15 most intense peptide precursor ions were subjected to fragmentation. The AGC for MS1 was set to 3×106 with a max injection time of 120 ms, the AGC for MS2 ions was set to 1×105 with a max injection time of 150 ms, and the dynamic exclusion was set to 90 s. Raw data analysis of LFQ experiments was performed by Fox Chase Cancer Center Biostatistics and Bioinformatics Facility.

Proteolytic peptides were resuspended in 0.1% formic acid and separated with a Thermo Scientific RSLC Ultimate 3000 on a Thermo Scientific Easy-Spray C18 PepMap 75 μm × 50 cm C-18 2 μm column. For MIB runs, a 240-minute gradient of 4%–25% acetonitrile with 0.1% formic acid was used. For total proteome runs, a 305-minute gradient of 2%–20% (180 minutes), 20%–28% (45 minutes), and 28%–48% (20 minutes) acetonitrile with 0.1% formic acid was used. Both gradients were run at 300 nL/minute at 50^oC. Eluted peptides were analyzed by Thermo Scientific Q Exactive or Q Exactive plus MS utilizing a top 15 methodology, in which the 15 most intense peptide precursor ions were subjected to fragmentation. The AGC for MS1 was set to 3 × 10⁶ with a max injection time of 120 minutes; the AGC for MS2 ions was set to 1 × 10⁵ with a max injection time of 150 minutes; and the dynamic exclusion was set to 90 seconds.