Chang Rachel Yoon Kyung, Etheridge Naomi, Dodd Peter R, Nouwens Amanda S
School of Chemistry and Molecular Biosciences, University of Queensland, Australia.
School of Chemistry and Molecular Biosciences, University of Queensland, Australia.
Neurochem Int. 2014 Sep;75:66-75. doi: 10.1016/j.neuint.2014.05.011. Epub 2014 Jun 2.
Brain tissue from Alzheimer's disease (AD) patients shows significant loss of synapses in selected regions. Synaptic degeneration is the best predictor for loss of cognitive functions ante mortem. The molecular mechanisms underlying this degeneration remain unknown. Our previous two-dimensional gel-electrophoresis proteomics study found that 26 synaptic proteins are differentially expressed in Alzheimer's brain. It is difficult to quantify global protein expression using this technique because (a) several proteins can migrate together and (b) isoforms of the same protein can migrate to different places. The present study estimated global synaptic protein levels by label-free multiple reaction monitoring. Multiple reaction monitoring is a powerful and sensitive mass spectrometry technique that specifically targets multiple protein of interests. The severely AD-affected hippocampus was compared with motor cortex, a relatively spared region. We targeted ten proteins in autopsy brain based on the earlier study. Analytes separated by high performance liquid-chromatography were monitored on a hybrid triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. With the use of an internal standard protein, linear and highly reproducible (CV<9%) label-free assays were achieved. Data were contrasted with the gel-based study to highlight differences and similarities. Significantly higher expression levels of peroxiredoxin-1 (may provide antioxidant protection) and dihydropyrimidinase-related protein-1 (associated with cytoskeletal remodeling) were found in AD hippocampus. Significantly lower levels of peroxiredoxin-1 and the energy-related enzymes creatine kinase B and fructose-bisphosphate aldolase C were found in non-AD hippocampus. Our previously reported difference in synaptotagmin expression is probably isoform-specific. These findings suggest potential roles of key proteins in synaptic loss in AD, and/or a protective mechanism in non-AD brain tissue.
阿尔茨海默病（AD）患者的脑组织在特定区域显示出明显的突触丧失。突触变性是生前认知功能丧失的最佳预测指标。这种变性背后的分子机制仍然未知。我们之前的二维凝胶电泳蛋白质组学研究发现，26种突触蛋白在阿尔茨海默病大脑中差异表达。使用这种技术难以量化整体蛋白质表达，因为（a）几种蛋白质可能一起迁移，以及（b）同一蛋白质的异构体可能迁移到不同位置。本研究通过无标记多反应监测估计整体突触蛋白水平。多反应监测是一种强大且灵敏的质谱技术，专门针对多种感兴趣的蛋白质。将严重受AD影响的海马体与相对未受影响的运动皮层进行比较。基于早期研究，我们在尸检大脑中靶向了十种蛋白质。通过高效液相色谱分离的分析物在混合三重四极杆线性离子阱质谱仪上以多反应监测模式进行监测。通过使用内标蛋白，实现了线性且高度可重复（CV<9%）的无标记分析。将数据与基于凝胶的研究进行对比，以突出差异和相似之处。在AD海马体中发现过氧化物酶体增殖物激活受体1（可能提供抗氧化保护）和二氢嘧啶酶相关蛋白1（与细胞骨架重塑相关）的表达水平显著更高。在非AD海马体中发现过氧化物酶体增殖物激活受体1以及能量相关酶肌酸激酶B和果糖二磷酸醛缩酶C的水平显著更低。我们之前报道的突触结合蛋白表达差异可能是异构体特异性的。这些发现提示关键蛋白在AD突触丧失中的潜在作用，和/或在非AD脑组织中的保护机制。
