高丰度蛋白去除后干燥综合征口腔液生物标志物候选物的鉴定
Identification of Sjögren's syndrome oral fluid biomarker candidates following high-abundance protein depletion.
作者信息
Deutsch Omer, Krief Guy, Konttinen Yrjö T, Zaks Batia, Wong David T, Aframian Doron J, Palmon Aaron
机构信息
Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel.
Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University of Jerusalem, Israel, Department of Medicine, Biomedicum Helsinki, Helsinki University Central Hospital, ORTON Orthopaedic Hospital, Helsinki, COXA Hospital for Joint Replacement, Tampere, Finland, School of Dentistry and Dental Research Institute, University of California at Los Angeles, Los Angeles, CA, USA and Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel.
出版信息
Rheumatology (Oxford). 2015 May;54(5):884-90. doi: 10.1093/rheumatology/keu405. Epub 2014 Oct 21.
OBJECTIVE
SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates.
METHODS
Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed.
RESULTS
The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS.
CONCLUSION
The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.
目的
干燥综合征是一种自身免疫性外分泌腺病，在美国约有100万患者受其影响，主要在中年女性中被诊断出来。口腔液体作为身体的一面镜子，被认为是一种理想的非侵入性诊断工具。此前我们开发了口腔液体高丰度蛋白的去除技术，以增加低丰度蛋白的可视化。因此，本研究的目的是检验去除预处理对干燥综合征口腔液体生物标志物候选物鉴定潜力的影响。
方法
从18名女性干燥综合征患者和18名年龄及性别匹配的健康对照者中收集非刺激性口腔液体。使用亲和及免疫去除方法去除高丰度蛋白，随后进行半定量二维凝胶电泳和定量二甲基化液相色谱串联质谱分析（LC-MS/MS）。为初步验证质谱结果，进行了蛋白质印迹分析。
结果
蛋白质组学分析前使用去除策略使鉴定能力提高了3倍。总体而言，鉴定出79种生物标志物候选物。折叠变化最显著的蛋白质与干燥综合征血清或组织因子有关。此外，对干燥综合征患者中增加超过3倍的蛋白质进行生物信息学分析，显示出钙结合蛋白、防御反应蛋白、参与凋亡调节的蛋白、应激反应蛋白和细胞运动相关蛋白。对肌动蛋白结合蛋白和碳酸酐酶I进行蛋白质印迹初步验证，显示出与定量质谱鉴定的相似表达谱趋势。
结论
新型口腔液体去除方法的重要性已得到明确证明，它不仅能提高生物标志物候选物的可见性，还能揭示该综合征可能涉及的机制。这对我们将口腔液体用作未来诊断液体的能力做出了重大贡献。
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