Pierce quantitative colorimetric peptide assay

The peptide concentrations of resuspended peptide samples were determined using the Pierce™ Quantitative Colorimetric Peptide Assay (cat number 23275, Thermofisher Scientific, Brisbane, Australia) according to the manufacturer’s instructions. An appropriate addition of iRT buffer was used to equalize all peptide concentrations prior to analysis by LC-MS/MS.

Whole EV pellets were previously flash-frozen after collection. EVs were processed for LC-MS/MS using a PreOmics iST Kit (P.O.00027). Briefly, EV pellets were brought up in 50 µl of provided LYSE solution and boiled with agitation for 10 min. The provided enzymes mixture (Trypsin and LysC) were resuspended in 210 µl of RESUSPEND buffer, mixed, and added to the lysed EVs. Samples were allowed to mix at 500 rpm for 1.5 hr at 37°C, before being quenched with 100 µl of STOP solution. Sample was spun in provided C18 spin cartridge and washed 1× with 200 µl of WASH 1 and WASH 2. Peptides were eluted with 2× 100 µl of ELUTE, dried, and resuspended with the provided LC-LOAD solution. Peptides were quantified using Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, 23275).

A 100 μg proteins from each sample were precipitated in 20% trichloroacetic acid (TCA) at 4°C. Protein pellets were washed with ice-cold acetone and re-suspended in 25 μl TEAB (100 mM) and 25 μl 2,2,2-trifluoroethanol (TFE). Proteins were reduced with 1 μl of tris(2-carboxyethyl)phosphine (TCEP, 500 mM), alkylated with iodoacetamide (IAA, 30 mM), and digested with 2.5 μg/sample of trypsin/lysC (Promega, Madison, WI, United States) overnight at 37°C. The digested peptides were quantified using the Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific, Waltham, MA, United States). 40 μg of peptides from each specific sample was labeled with the Thermo Scientific TMTsixplex Isobaric Mass Tagging Kit (JSC-E1 (ground 1) with TMT⁶-128, JSC-E2 (ground 2) with TMT⁶-130, JSC-S1 (ISS 1) with TMT⁶-129, JSC-S2 (ISS 2) with TMT⁶-131) according to the manufacturer’s protocol. All labeled-peptide mixtures were combined into a single tube, mixed, and fractionated using the Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit. While this kit usually uses eight fractions with step elution of up to 50% acetonitrile, ninth fraction was added eluting at 100% acetonitrile. Nine fractionated samples were dried using a SpeedVac concentrator and re-suspended in 1% (v/v) formic acid prior to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

Cells were cultured in T75 flasks in triplicates (three flasks per cell line), and the seeding density was 600,000 cells per flask. Proteins were harvested at 90% confluency, and 72 h after seeding, cells were washed twice with ice-cold PBS and lysed by 450 μl lysis buffer containing 4% sodium dodecyl sulfate (SDS, MP Biomedicals) in 100 mM Tris (Sigma). Flasks were kept on ice for 10 min. The cell lysates were transferred to 1.5 ml Eppendorf tubes. After five freeze (−80 °C)/thaw (room temperature) cycles, the sample was spun at 20,718g for 20 min at 4 °C. The supernatant was collected and aliquoted in new tubes and stored at −80 °C. Protein quantification was measured with BCA protein assay (Pierce).
For Filter-Aided Sample Preparation (FASP), an equivalent of 300 μg of proteins in 150 μl from each sample was reduced with 100 mM dithiothreitol (DTT), and samples were then processed using FASP protocol (24 (link)). Proteins on the filters were digested twice at 30 °C with trypsin (enzyme-to-substrate ratio: 1:100 (w/w); 3 μg × 2), first overnight and then for another 6 h in a final volume of 200 μl. The resulting peptides were desalted using a C18 solid-phase extraction cartridge (Empore, Agilent technologies). Peptides were resuspended in 50 μl 1% formic acid and quantified using pierce quantitative colorimetric peptide assay (product 23275, Thermo Scientific).

The Pierce Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, Massachusetts, USA) was used to determine peptide concentrations post on-bead digestion. 20 µg of tryptic digest pooled from biological samples, were analysed using HpHRP. This was performed using a Dionex UltiMate 3000 RSLC (Thermo Fisher Scientific, Massachusetts, USA) coupled with an Acclaim PA II column (1.0 mm × 15 cm, C18, 3 µm, 120 Å) (Thermo Fisher Scientific, Massachusetts, USA). Mobile phase A contained 20 mM ammonium hydroxide in water (pH 9.6) and Mobile phase B contained 20 mM ammonium hydroxide (pH 9.6) supplemented with 80% acetonitrile in water. Peptide separation was done at 50 µl/min using a 35-min gradient (4–40% B). 30 fractions were collected every 30 secs from 12.5 to 27.5 min and then pooled to generate 10 concatenated fractions as per the following pooling scheme: (F1 = [1, 11, 21]; F2 = [2, 12, 22]; F3 = [3, 13, 23]; F4 = [4, 14, 24]; F5 = [5, 15, 25]; F6 = [6, 16, 26]; F7 = [7, 17, 27]; F8 = [8, 18, 28]; F9 = [9, 19, 29], F10 = [10, 20, 30]). Pooled fractions were vacuum dried using a CentriVap (Labconco, Missouri, USA) and stored at − 80 °C until further analysis.