Top-down proteomic identification of protein biomarkers of Xylella fastidiosa subsp. fastidiosa using MALDI-TOF-TOF-MS and MS/MS

Abstract

Protein biomarkers were identified from bacterial cell lysates of two sequenced strains of Xylella fastidiosa subsp. fastidiosa (Xff), the causal agent of Pierce's Disease of grapevine, using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. Proteins were identified from their intact masses as well as sequence-specific fragment ions resulting from the aspartic acid effect detected by MS/MS and post-source decay (PSD). Two Xff strains from California were studied, M23 isolated from almond in Kern County and Stag's Leap isolated from grapevine in Napa County. The two strains are pathogenically identical or highly similar. Most of the proteins identified were highly conserved, e.g., 10 kDa chaperonin, cold-shock and/or DNA-binding, ribosomal and hypothetical proteins. The amino acid sequences and post translational modifications (PTM) of these conserved proteins were identical between the two Xff strains. However, the amino acid sequence of an outer membrane/hypothetical protein differed significantly between the two strains and could be a useful biomarker to distinguish between these closely related strains. In silico signal peptide analysis suggest that the outer membrane/hypothetical protein are secreted lipoproteins (SPII) with a putative prosthetic group attached to the only cysteine residue (C19) before or after removal of an 18-residue N-terminal signal peptide. However, only y-type fragment ions were detected by MS/MS-PSD which precluded confirmation of the site of sequence truncation and/or attached PTM. The annotated genomes of the two strains indicate ten to twenty proteins that are either lipoproteins or are associated with assembly, modification, or transport of lipoproteins. In consequence, a variety of prosthetic groups, e.g. diacylglycerol, may be attached to C19 in the mature protein.