A) Workflow describing the procedures used to isolate X. fastidiosa Temecula1 outer membrane proteins (OMPs), outer membrane vesicles (OMVs), surface peptides (surfaceome) and soluble supernatant proteins (SSPs). Outer membrane proteins were extracted using 0.1 M sodium carbonate buffer (pH 11.0) followed by mass spectrometry. To isolate surface peptides, cells (4 × 10 8 cells/mL) were harvested from a four- to six- day-old culture and subjected to tryptic digestion (cell shaving). Peptides were concentrated (5×) and subjected directly to LC/MSMS. OMVs were purified from the culture supernatant using two ultracentrifugation steps (38,000 × g for 1 h to pellet cell debris followed by 150,000 × g for 3 h for OMVs precipitation). The remaining supernatant was concentrated ( 75 to 100×) using Amicon Ultra-15 3 K filter units to identify SSPs by mass spectrometry. ( ∼ B) SDS-PAGE 12% resolution of 10 μg Xff total protein (TP), OMP ( Table S2) and SSPs ( Table 1). Three putative lipase/esterases (PD1703, PD1702, and PD1211) were identified in the highlighted band, which was composed mostly of PD1703 (LesA) and PD1702. ( C) Venn diagram quantifying the number of proteins identified in each subcellular proteome. The major outer membrane protein MopB was found in all samples, although it was very poorly represented in the secretome. The lipase/esterase LesA was found in the secretome, surfaceome and OMV proteome.