Toby T K, Abecassis M, Kim K, Thomas P M, Fellers R T, LeDuc R D, Kelleher N L, Demetris J, Levitsky J
Department of Molecular Biosciences and Chemistry, Northwestern University, Chicago, IL.
Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL.
Am J Transplant. 2017 Sep;17(9):2458-2467. doi: 10.1111/ajt.14359. Epub 2017 Jun 27.
Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent "top-down" proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight-based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.
肝移植受者急性排斥反应的生物标志物谱可改善对受者的诊断和管理。我们的目标是使用一种新兴的“自上而下”蛋白质组学方法，确定循环免疫细胞中急性排斥反应的诊断性蛋白质异构体特征。我们通过基于分子量的分级分离，从26名非病毒性肝移植受者中制备了经过不同处理和冷冻保存的细胞裂解物，并分三步对全蛋白进行质谱分析：（i）纳升毛细管液相色谱与高分辨率串联质谱联用；（ii）数据库搜索以鉴定和表征完整的蛋白质异构体；（iii）通过与研究设计相匹配的分层线性模型进行数据处理，以量化排斥反应患者与肝功能正常患者以及无排斥反应的急性功能障碍患者之间蛋白质异构体的倍数变化。与正常和非特异性对照相比，排斥反应患者中出现了差异表达的蛋白质异构体，在传统富含血清的培养基中储存的细胞制剂中最为明显。通过基因本体论和通路分析工具将这些蛋白质映射回基因的分析揭示了多种信号通路，包括由细胞因子和趋化因子介导的炎症。需要进行更大规模的研究来验证这些新的排斥反应特征，并测试它们在临床管理中的预测价值。
