Trypsin v511

Three biological experiments were carried out with three technical replicates. The total number of samples analyzed by MALDI was 300. The number of technical replicates for protein identification by MALDI mass spectrometry was 2-3 (up to 5 for important and low-abundance proteins). Individual protein spots, selected on the basis of image-analysis output, were excised and digested in-gel with trypsin (Trypsin V511, Promega, Madison, WI, USA) as previously described [19 (link)]. For MALDI-TOF identification, 0.5–1 μL of the sample (50% solution of acetonitrile in water, 0.1% TFA) was placed on a ground steel MALDI target plate or AnchorChip or SmallAnchor (depending on the protein quantity), and 0.5–1 μL of the matrix (α-cyano-4-hydroxycinnamic acid) (Bruker Daltonics, Bremen, Germany) was added.

The EVs derived from calli were treated with Proteinase K (20 µg/mL) for 1 h at 37 °C, with intermittent gentle mixing every 10 min to ensure uniform exposure of the vesicles to the enzyme. Following Proteinase K treatment, the proteolytic activity was curtailed by introducing 5% PMSF into the samples. The subsequent inhibition process involved incubation for 10 min at room temperature. Further inactivation of the proteinase was achieved by subjecting the samples to a heat treatment at 90 °C for 5 min. The extraction of total proteins followed established protocols, as previously described [77 (link)]. Protein extracts obtained from the EV samples were separated by 12.5% SDS-PAGE and visualized by Coomassie staining. Upon completion of electrophoresis, gel slices containing the protein components of interest were meticulously excised into smaller fragments. The excised gel slices were subjected to in-gel digestion with trypsin (Trypsin V511; Promega, Madison, WI, USA) using a previously described procedure [83 (link)]. Following trypsin digestion, the resulting peptide mixture was dried using a Concentrator plus (Eppendorf, Hamburg, Germany).

The total number of samples analyzed by MALDI was 203. The number of technical replicates was 2–3 (up to 8 for important proteins). Individual protein spots selected on the basis of image-analysis output were excised and digested in-gel with trypsin (Trypsin V511, Promega, Madison, WI, USA) as previously described^{30 (link)}. For MALDI-TOF identification, 0.5–1 μl of the sample (50% solution of acetonitrile in water, 0.1% TFA) was placed on a ground steel MALDI target plate or AnchorChip or SmallAnchor (depending on the protein quantity; also see Supplementary Dataset S1), and 0.5–1 μl of the matrix (α-cyano-4-hydroxycinnamic acid) was added. For LC-ESI-MS/MS, 10-μl protein samples dissolved in water containing 0.1% TFA were used.