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

Authors: Fagerquist, Clifton - Keith; Wallis, Christopher; Chen, Jianchi

Submitted to: International Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/2023
Publication Date: 4/13/2023
Citation: Fagerquist, C.K., Wallis, C.M., Chen, J. 2023. Top-down proteomic identification of protein biomarkers of Xylella fastidiosa subsp. fastidiosa using MALDI-TOF-TOF-MS and MS/MS. International Journal of Mass Spectrometry. 489. Article 117051. https://doi.org/10.1016/j.ijms.2023.117051.
DOI: https://doi.org/10.1016/j.ijms.2023.117051

Interpretive Summary: Xylella fastidiosa subsp. fastidiosa (Xff) is a bacterium that causes Pierce’s Disease of grapevine that is highly destructive in viticulture. Xff is transmitted by sharpshooter leafhoppers (e.g. Glassy-winged sharpshooter, blue-green sharpshooter, etc.). Because of the detrimental effects of this insect-transmitted pathogen on many different agriculturally important plants, a number of Xff strains have been analyzed using genomic and proteomic technologies to better understand their virulence and pathogenicity. The ultimate goal of these investigations is to develop interventions to disrupt the insect vectors carrying the bacterium or to enable plants to better resist a bacterial infection when it occurs. The protein biomarkers of two Xff strains (M23 and Stag’s Leap isolated in Kern and Napa, California counties, respectively) were analyzed using MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis. Highly conserved proteins (chaperone, DNA-binding, cold-shock, ribosomal proteins, etc.) having identical amino acid sequences were identified from both Xff strains. However, an outer membrane/hypothetical protein showed significant differences in sequence between the two strains making it useful as a biomarker to distinguish the generally highly similar Xff strains and to facilitate understanding of Xff biology.

Technical 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.