Liquid chromatography–mass spectrometry

Liquid chromatography-mass spectrometry of conditioned medium (CM) samples

Cells were seeded in 10-cm plates, and serum-free medium was added 24 h later. CM samples were collected 16 h later, filtered through 0.45 µm filter, and concentrated consecutively using Amicon Ultra-15 10K and Ultra-0.5 10K centrifugal filters (Sigma). Proteins were solubilized with 25uL of 5% SDS, 50mM TEAB, pH 7.55 followed by a room temperature incubation for 30 minutes. The supernatant containing the proteins of interest was then transferred to a new tube, reduced by making the solution 10mM Tris(2-carboxyethyl)phosphine (TCEP) (Thermo, #77720), and further incubated at 65C for 10 minutes. The sample was then cooled to room temperature and 2 uL of 0.5M iodoacetamide acid was added and allowed to react for 20 minutes in the dark after which 0.5 mL of 2M DTT was added to quench the reaction. Then, 5 mL of 12% phosphoric acid was then added to the 50 uL protein solution followed by 350 uL of binding buffer (90% Methanol, 100mM TEAB final; pH 7.1). The resulting solution was administered to an S-Trap spin column (Protifi, Farmingdale NY) and passed through the column using a bench top centrifuge (30 s spin at 4,000 g). The spin column was then washed three times with 400 mLof binding buffer and centrifuged (1200rpm, 1min). Trypsin (Promega, #V5280, Madison, WI) was then added to the protein mixture in a ratio of 1:25 in 50mM TEAB, pH = 8, and incubated at 37C for 4 hours. Peptides were eluted with 80uL of 50mM TEAB, followed by 80 mL of 0.2% formic acid, and finally 80 mL of 50% acetonitrile, 0.2% formic acid. The combined peptide solution was then dried in a speed vacuum (room temperature, 1.5 hours) and resuspended in 2% acetonitrile, 0.1% formic acid, 97.9% water and aliquoted into an autosampler vial.

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