Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum.
Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum.
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DOI：
10.1186/s12014-017-9156-y
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发表时间：
2017
影响因子：
3.8
通讯作者：
Kruh-Garcia NA
中科院分区：
文献类型：
--
作者：
Mehaffy C;Dobos KM;Nahid P;Kruh-Garcia NA
结核分枝杆菌 (Mtb) 是结核病 (TB) 的病原体，而结核病是传染病导致死亡的第一大原因。结核病诊断是通过细菌 DNA 的显微镜检查、培养或 PCR 扩增来进行的，所有这些都需要患者痰液或受感染组织的活检。检测血清中的分枝杆菌产物作为诊断或疾病状态的生物标志物将改进现有方法。由于血清中分枝杆菌产物的丰度较低，我们探索了外泌体富集以提高敏感性。 Mtb 存在于细胞内，其分泌的蛋白质已被证明被包装到宿主外泌体中并释放到血流中。外泌体可以很容易地纯化，确保从宿主血清蛋白的复杂混合物中富集分枝杆菌分析物。对多反应监测测定进行了优化，以增强对从结核病患者血清中纯化的外泌体中 41 种 Mtb 肽的检测。从健康个体血清中分离出的外泌体用于创建和验证独特的数据分析算法并识别过滤器以降低由于宿主 m/z 干扰而导致的误报率。最终优化的方法在来自 TB 阳性患者的 40 份外泌体样本中进行了测试。我们的增强方法提供低飞摩尔范围内的检测限和定量平均，用于检测血清中的分枝杆菌产物。 92.5% 的结核病阳性患者中至少鉴定出一种分枝杆菌肽。来自 Mtb 蛋白的四种肽（Cfp2、Mpt32、Mpt64 和 BfrB）显示，与健康对照相比，活动性结核病个体的归一化总峰面积显着更高；这些蛋白质中的三种肽此前并未与活动性结核病患者的血清外泌体相关。一些检测到的肽与特定地理位置显着相关，突出显示了可以与每个给定区域内循环的 Mtb 菌株相关的潜在标记。展示了一种检测人血清外泌体中超低丰度 Mtb 肽的增强 MRM 方法，突显了该方法用于结核病诊断生物标志物开发的潜力。本文的在线版本 (doi:10.1186/s12014-017-9156-y) 包含补充材料，可供授权用户使用。
Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m/z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development. The online version of this article (doi:10.1186/s12014-017-9156-y) contains supplementary material, which is available to authorized users.