Dionex ultimate 3000 rslc nano uplc system

A total
of six protein–DON pellet fractions were retrieved per DON
coating condition and pooled in pairs for analysis. Samples were separated
by SDS-PAGE on a 12% polyacrylamide gel and stained with Coomassie
blue. Each gel lane was entirely sliced and subjected to in-gel digestion.
The gel pieces were washed twice with 50% ethanol in 50 mM ammonium
bicarbonate (AB, Sigma-Aldrich) for 20 min and dried by vacuum centrifugation.
Proteins were reduced with 10 mM dithioerythritol (Merck-Millipore)
for 1 h at 56 °C followed by a washing-drying step, as described
above. Reduced proteins were alkylated with 55 mM iodoacetamide (Sigma-Aldrich)
for 45 min at 37 °C in the dark followed by a washing-drying
step, as described above. Proteins were digested overnight at 37 °C
using mass spectrometry grade Trypsin Gold (Trypsin Gold, Promega)
at a concentration of 12.5 ng/μL in 50 mM AB supplemented with
10 mM CaCl₂. Resulting peptides were extracted in 70% ethanol,
5% formic acid (Merck-Millipore) twice for 20 min, dried by vacuum
centrifugation. Resulting peptides were desalted on StageTips^{31 (link)} and dried under a vacuum concentrator. For liquid
chromatography and mass spectrometry (LC–MS)/MS analysis, resuspended
peptides were separated by reversed phase chromatography on a Dionex
Ultimate 3000 RSLC nano UPLC system in-line connected to an Exploris
480 Orbitrap mass spectrometer (Thermo Fisher Scientific).

To facilitate the identification of the low abundance proteins, both lymphatic exudate and plasma were IgG- and albumin-depleted with Aurum Serum Spin Colums (Bio-Rad) as previously described (Clement et al., 2013 (link)). EV fractions were analyzed without prior depletion. 4 µg of total protein was subjected to an “in solution” tryptic digest. 2 µg of each trypsinized sample was used for the nanoLC-MS/MS analysis on an Orbitrap Elite mass spectrometer. Prior to the injection into the mass spectrometer, peptides were desalted on C18 stageTips (Rappsilber et al., 2007 (link)), dried down by vacuum centrifugation, and separated by reversed phase chromatography on a Dionex Ultimate 3000 RSLCnano UPLC system connected in-line with an Orbitrap Elite (Thermo Fisher Scientific).