Protein preparation and mass spectrometry analysis

Xff SSPs and OMV proteins were resolved by SDS-PAGE prior to in-gel digestion to reduce the amount of non-protein contaminants in the samples. Peptides from the cell shaving (surfaceome) procedure were desalted using Aspire RP30 desalting tips (Thermo-Fisher Scientific) and subjected directly to LC/MSMS analysis. For the in-gel digestion used for SSPs and OMV proteome analysis, gel pieces were washed twice with 100 to 150 μL 50 mM ammonium bicarbonate (AmBic; pH 8.0), followed by dehydration with acetonitrile (ACN; three to four times the total volume of gel pieces) for 10 to 15 min. Proteins were reduced for 30 min at 56°C in a solution of 10 mM DTT and 50 mM AmBic. Gel pieces were dehydrated again, followed by replacement of ACN by 55 mM iodoacetamide in 50 mM AmBic. Gel pieces were incubated 20 min in the dark at room temperature, followed by two washes with 150 to 200 μL of 50 mM AmBic for 15 min each. Gel pieces were dehydrated with ACN, dried by speed vacuum centrifugation and subjected to tryptic digestion overnight. Peptides were extracted by adding 60% ACN and 0.1% trifluoroacetic acid (TFA) in water to the gel pieces, followed by sonication for 10 min. The solution containing the peptides was mixed with the supernatant resulting from the tryptic digestion, followed by speed vacuum centrifugation. Digested peptides were then desalted using Aspire RP30 desalting tips and resuspended in loading buffer.

The digested peptides were analyzed using a QExactive mass spectrometer (Thermo Fisher Scientific) coupled with an Easy-LC (Thermo Fisher Scientific) and a nanospray ionization source. The peptides were loaded onto a trap (100 micron, C18 100 Å 5U) and desalted online before separation using a reverse phased column (75 micron, C18 200 Å 3U). The gradient duration for separation of peptides was 60 min using 0.1% formic acid and 100% ACN for solvents A and B respectively. Data was acquired using a data-dependent ms/ms method with a full scan range of 300 to 1600 Da and a resolution of 70,000. The ms/ms method’s resolution was 17,500 with an isolation width of 2 m/z with normalized collision energy of 27. The nanospray source was operated using 2.2 KV spray voltage and a heated transfer capillary temperature of 250°C. Raw data was analyzed using X!Tandem and visualized using Scaffold Proteome Software (Version 3.01). Samples were searched against Uniprot databases appended with the cRAP database, which recognizes common laboratory contaminants. Reverse decoy databases were also applied to the database prior to the X!Tandem searches.

For the grapevine proteomic analysis, leaf proteins were precipitated using a ProteoExtract protein precipitation kit (Calbiochem) followed by dehydration overnight in a sterile fume hood. The protein pellet was resuspended in 50 mM AmBic (pH 8.0) and subjected to an in-solution tryptic digestion. Digested peptides were then desalted and subjected to LC/MSMS as described above.