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Comparative Global Protein Profiling of Pediatric Sinonasal Secretions from Healthy and Chronic Rhinosinusitis Patients

机译：健康和慢性鼻-鼻窦炎患者小儿鼻窦分泌物的全球蛋白质比较

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摘要

Chronic rhinosinusitis (CRS) is a highly prevalent disease of the upper respiratory tract and is characterized by mucous overproduction and hyperplasia of the submucosal glands in the sinus mucosa. The composition of sinonasal secretions, however, is minimally characterized. In this study, proteomic analyses were performed to identify the protein components, including specific mucin glycoproteins (mucins), in the sinonasal secretions of control and CRS pediatric patients. Secretions were collected at Children's National Medical Center (CNMC) from (a) CRS patients undergoing functional endoscopic sinus surgery and (b) control patients without CRS undergoing surgical procedures un-related to CRS. Secretions were processed for proteomic analysis using established protocols in the CNMC Proteomics Core. Proteins were separated by electrophoresis on SDS-polyacrylamide gels, bands were excised, digested with trypsin, and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins were identified using the Sequest algorithm in the Bioworks Browser Software against the UniProt database. Fold-change significance was calculated using the QSpec algorithm. Western blot analysis was performed to validate proteomic findings and to determine whether MUC5B, a glandular mucin, was semi-quantitatively increased in CRS sinonasal secretions.;Five samples from CRS patients and three from non-CRS patients have been analyzed by proteomic profiling to date. A total of 294 unique proteins were identified across all samples, some of which were common to both CRS and control samples and others that were differentially expressed, including proteins previously reported to be associated with CRS [BP1 fold-containing family A member 1 (BPIFA1), MUC5B, and chitinase-3-like protein 1 (CHI3L1)]. The protein with the most significant negative fold-change (decreased abundance in CRS samples) was retinal dehydrogenase 1 (ALDH1A1, log2 fold-change = -4.39). The protein with the highest significant positive fold-change in CRS samples was BPIFA1 (log2 fold-change = 3.93). Glycoprotein-340, BPIFB1, S100-A9, and serpin A1 were validated by western blot analyses and showed overall increased expression in CRS samples compared to control samples. Proteomic analyses identified MUC5B mucin in all CRS samples and in two control samples. MUC5AC mucin was identified in three CRS samples and two control samples. The relative abundance based on spectral count data of MUC5B compared to MUC5AC in the CRS samples was significantly higher for MUC5B (p < 0.05). Based on western blot analyses, there was no significant increase in the proportion of MUC5B to MUC5AC expression in CRS samples compared to controls, although there was a trend towards a higher ratio of MUC5B to MUC5AC. Data analysis using protein knowledge database tools revealed that proteins showing the most significant expression in CRS samples compared to non-diseased samples were involved in biological processes such as biological regulation, cellular processes, response to stimuli, and immune system response. Proteomic data suggest that MUC5B is significantly more abundant than MUC5AC in CRS patients, likely reflecting the increased glandular hyperplasia in these patients. Further investigation by proteomic analysis and validation by western blot analysis of proteins present in CRS secretions may provide more insight into the pathogenesis of CRS in pediatric patients.
机译：慢性鼻-鼻窦炎（CRS）是上呼吸道的一种高度流行的疾病，其特征是鼻窦粘膜的粘液过度产生和粘膜下腺的增生。然而，鼻窦分泌物的组成很少。在这项研究中，进行了蛋白质组学分析，以识别对照组和CRS小儿鼻窦分泌物中的蛋白质成分，包括特定的黏蛋白糖蛋白（黏蛋白）。在儿童国家医学中心（CNMC）收集分泌物，这些分泌物是（a）接受功能性内窥镜鼻窦手术的CRS患者和（b）没有接受CRS的与CRS无关的外科手术的对照患者。使用CNMC蛋白质组学核心中已建立的协议对分泌物进行蛋白质组学分析。通过在SDS-聚丙烯酰胺凝胶上的电泳分离蛋白质，切下条带，用胰蛋白酶消化，并使用液相色谱串联质谱法（LC-MS / MS）进行分析。使用Bioworks Browser软件中的Sequest算法针对UniProt数据库鉴定蛋白质。使用QSpec算法计算倍数变化的重要性。进行蛋白质印迹分析以验证蛋白质组学发现并确定CUC鼻窦分泌物中MUC5B（腺黏液）是否半定量增加。迄今为止，已通过蛋白质组分析对CRS患者的五个样本和非CRS患者的三个样本进行了分析。在所有样品中共鉴定出294种独特蛋白，其中有些是CRS和对照样品共有的，还有一些差异表达，包括先前报道与CRS相关的蛋白[含BP1折叠家族A成员1（BPIFA1 ），MUC5B和几丁质酶3样蛋白1（CHI3L1）]。负倍数变化最大（在CRS样品中丰度降低）的蛋白是视网膜脱氢酶1（ALDH1A1，log2倍数变化= -4.39）。 CRS样品中具有最高显着正折叠倍数的蛋白质是BPIFA1（log2折叠倍数= 3.93）。糖蛋白340，BPIFB1，S100-A9和丝氨酸蛋白酶抑制剂A1已通过蛋白质印迹分析进行了验证，与对照样品相比，CRS样品中的表达总体增加。蛋白质组学分析确定了所有CRS样品和两个对照样品中的MUC5B粘蛋白。在三个CRS样品和两个对照样品中鉴定出MUC5AC粘蛋白。在CRS样品中，基于MUC5B的光谱计数数据相对于MUC5AC的相对丰度明显高于MUC5B（p <0.05）。根据蛋白质印迹分析，与对照组相比，CRS样品中MUC5B与MUC5AC表达的比例没有显着增加，尽管MUC5B与MUC5AC的比例有增加的趋势。使用蛋白质知识数据库工具进行的数据分析显示，与未患病样品相比，在CRS样品中表现出最显着表达的蛋白质参与了生物过程，例如生物调节，细胞过程，对刺激的反应以及免疫系统反应。蛋白质组学数据表明，CRS患者中MUC5B的含量明显高于MUC5AC，这可能反映了这些患者中腺体增生的增加。通过蛋白质组学分析进行进一步研究，并通过蛋白质印迹分析对CRS分泌物中存在的蛋白质进行验证，可能会为儿童患者CRS的发病机理提供更多见解。

著录项

  • 作者

    Saieg, Amarel.;

  • 作者单位

    The George Washington University.;

  • 授予单位 The George Washington University.;
  • 学科 Biochemistry.
  • 学位 M.S.
  • 年度 2013
  • 页码 155 p.
  • 总页数 155
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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