Mass spectrometry raw data and ion library

This dataset consists of raw mass spectrometry files of EVs isolated using EXO-NET from human plasma.

Filter Aided Sample Preparation: For SWATH analysis, individual exosome samples were processed using the Filter Aided Sample Preparation (FASP) method [26]. A total of 15 µg of exosome protein from each sample was reduced with equal volume of lysis buffer containing 8% SDS, 100 mM Tris, pH 7.6 and 0.2 M DTT, followed sonication and heating of samples at 95°C, each. Samples were allowed to cool down completely before adding 8 M urea in 100 mM Tris, pH 8.5. Samples were transferred into a Nanosep® filter unit with a 30K molecular weight cut off and centrifuged for 10,000 g for 15 min. Then, filter units were washed with 400 µl of urea buffer and centrifuged for 10,000 g for 15 min. Samples were alkylated by addition of 100 µl of 50mM IAA in 8M urea buffer and incubated in the dark for 20 min. The filter units were washed with 8M urea buffer followed by of ABC. Proteins were digested using 0.3 µg of trypsin and incubated overnight at 37°C.

Analysis of peptides: Tryptic digest was loaded onto a reversed phase trap column (CHROMXP C18CL 5um, 10 x 0.3mm; Eksigent, Redwood City) and on column wash was performed for 15 min (3 ul/min) followed by peptide separation on reversed phase CHROMXP C18CL 3 um, 120 A0, 150 x 0.075mm; (Eksigent, Redwood City) analytical column. LC gradient started with 95% mobile phase A (H2O/ 0.1% FA), 5% B (ACN/ 0.1% FA) at 0 min and increase to 10% B over for 2 min and then a 58-min linear gradient to 40% B followed by 50% B for 5 min. Mobile phase B was then increased from 50% to 95 % over 10 min followed by column wash at 95% B for 15 min and re-equilibrated with 5% Buffer B for 6 min. Flow rate was kept at 250 nl/min during entire LC run. The resulting peptide samples were processed in IDA on an AB Sciex 5600 TripleTOF mass spectrometer with the top 18 precursor ions automatically selected for fragmentation. The data obtained were combined to establish a peptide ion database. For SWATH acquisition, the TripleTOF® 5600 System was configured as described by Gillet at al. [27]. Using an isolation width of 26 Da (25 Da of optimal ion transmission efficiency and 1 Da for the window overlap), a set of 32 overlapping windows was constructed covering the mass range 400 to 1200 m/z.