Development of Multiple Reaction Monitoring (MRM) assays to identify Brucella abortus proteins in the serum of humans and livestock

Brucellosis is important yet neglected zoonotic disease with a considerable public health concern. Despite high endemicity rates in the country, most cases of brucella are underreported due to the lack of diagnostic accuracy of existing assays. Despite considerable advancement in mass spectrometry-based approaches, the utility of these tools in the diagnosis of zoonotic diseases is mainly scarce. In the present study, a targeted Multiple Reaction Monitoring-based mass spectrometry approach (MRM-MS) 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 (B. abortus) proteins were generated. MRM -MS assays for the accurate detection of these peptides were optimized using117 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. Our present study provides the first proof of principle evidence of developing an MRM-based mass spectrometry approach with diagnostic utility for brucellosis.

布鲁氏菌病是一种危害严重却被忽视的人畜共患病，对公共卫生构成显著威胁。尽管该国布鲁氏菌病流行率居高不下，但由于现有检测方法的诊断准确性不足，多数病例漏报。尽管基于质谱的检测技术已取得长足进展，但这类工具在人畜共患病诊断中的应用仍较为匮乏。本研究开发了一种基于多反应监测的质谱方法（MRM-MS），用于筛选并鉴定人类与畜禽布鲁氏菌病的蛋白质生物标志物。研究通过对布鲁氏菌蛋白质组生成虚拟胰蛋白酶酶解肽段，完成蛋白特征肽段的筛选。经生物信息学分析，共获得对应10种流产布鲁氏菌（Brucella abortus, B. abortus）免疫显性蛋白的30条合成肽段。利用117份分层后的人类与畜禽血清样本（其中临床确诊样本45份、疑似样本62份、对照样本10份），本研究优化了可精准检测上述肽段的MRM-MS检测方法。通过高通量MRM检测，在多份临床确诊及疑似的人类与畜禽血清样本中，鉴定出4条肽段的特征离子跃迁。本研究首次提供了原理验证证据，证明基于MRM的质谱技术可用于布鲁氏菌病的诊断。