Proteomic Analysis of Lymphatic Exudate and Plasma

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).