Development of multiple reaction monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock.

ABSTRACT

In the present study, a targeted multiple reaction monitoring-mass spectrometry (MRM-MS) approach was developed to screen and identify protein biomarkers for brucellosis in humans and livestock. The selection of proteotypic peptides was carried out by generating in silico tryptic peptides of the Brucella proteome. Using bioinformatics analysis, 30 synthetic peptides corresponding to 10 immunodominant Brucella abortus proteins were generated. MRM-MS assays for the accurate detection of these peptides were optimized using 117 serum samples of human and livestock stratified as clinically confirmed (45), suspected (62), and control (10). Using high throughput MRM assays, transitions for four peptides were identified in several clinically confirmed and suspected human and livestock serum samples. Of these, peptide NAIYDVVTR corresponding to B. abortus proteins BruAb2_0537 was consistently detected in the clinically confirmed serum samples of both humans and livestock with 100% specificity. To conclude, a high throughput MRM-MS-based protocol for detecting endogenous B. abortus peptides in serum samples of humans and livestock was developed. The developed protocol will help design sensitive assays to accurately diagnose brucellosis in humans and livestock. The data associated with this study are deposited in Panorama Public (https//panoramaweb.org/rNOZCy.url with ProteomeXchange ID PXD034407).