珠联璧合：APT蛋白质组学助力客户分析揭示更年期血浆蛋白质谱的表达变化
2019-01-16
中科新生命
2957Proteomics in plasma of ovariectomized rats and those exposed to estradiol valerate. J Steroid Biochem Mol Biol. 2017. (IF=4.561)
研究背景：围绝经期是女性一生必经的生理过程，由于此阶段卵巢功能衰退，女性激素大幅度波动且逐渐下降，易使女性出现生理和心理功能失调，进而引发一系列相关症状和疾病。本文利用蛋白质组学的技术，首次全面分析了大鼠去卵巢后血浆蛋白质的变化情况，以及雌激素的治疗作用。这有助于更好地了解机体在围绝经期发生的变化的分子机制，并为围绝经期相关症状和疾病诊断生物标志物的挖掘和临床治疗方案的选择提供有价值的线索。
样本来源：雌性大鼠（Female Sprague-Dawley rat）血浆
技术路线： TMT蛋白质组+PRM验证
研究结果:
1、采用TMT蛋白质组分析去卵巢处理以及雌激素替代治疗处理对血浆中蛋白质组的影响，共检测到6528条肽段；其中与对照组（SHAM）相比，去卵巢组（OVX）中121个蛋白表达发生变化，36个上调，85个下调；与去卵巢组（OVX）相比，雌激素治疗组（OVX+E）引起117个蛋白差异表达，69个增加，48个降低；与对照组（SHAM）相比，雌激素治疗组（OVX+E）中109个蛋白表达发生变化，41个上调，68个下调；应用“优化筛选标准”，即差异仅发生于OVX/SHAM和OVX+E/OVX中且变化趋势相反，在OVX+E/SHAM中不表现出差异，对上述蛋白进一步筛选产生最优差异表达蛋白质共35个，15个上调，20个下调。
Table.1. Optimized differentially expressed proteins between groups
2、选取其中5个重要的差异表达蛋白质（GHR，LIFR，apoA IV，RTN和Lin28b）经PRM平行反应监测进一步得到验证，证明质谱结果可靠。
Figure 2. PRM results indicate excellent stability of the MS signal. Quantitative results of the heavy isotope-labeled peptide fragment in 9 samples are shown. It could be seen that the RSD differences of three fragment ions of the internal standard peptide fragment in 9 samples were all within 11%, indicating excellent stability of the MS signal.
3、生物信息学分析对鉴定到的差异血浆蛋白功能进行了聚类分析，发现这些差异表达的蛋白在组间可以被很好的分离，证明样品的选择和处理具有一定的代表性。
Figure 3. Differentially expressed proteins screened out were reasonable and accurate. Cluster Analysis. Through horizontal comparison, samples could be classified into three categories，suggesting that the selected differentially expressed proteins could effectively distinguish samples. Vertical comparison indicated that proteins could be classified into two categories with opposite directional variation, demonstrating the rationality of the selected differentially expressed proteins.
4、生物信息学分析对鉴定到的差异血浆蛋白功能进行了生物信息学注释（GO功能分析），发现这些差异表达的蛋白在分子功能、细胞组分和生物学过程方面分别主要被注释为蛋白结合、细胞和单有机体过程。
Figure 4. Differentially expressed proteins are mainly annotated as protein binding, cell, and single organism process.Gene ontology annotation. The differentially expressed proteins are mainly annotated as protein binding, cell, and single organism process in terms of molecular function, cell composition, and biological process, respectively.
5、生物信息学分析对鉴定到的差异血浆蛋白功能进行了生物信息学注释（KEGG功能分析），发现这些差异表达的蛋白在主要参与PI3-Akt生物信号通路。
Figure 5. The PI3-Akt pathway has the highest aggregation degree of differentially expressed proteins.KEGG Pathway Analysis. Change in estrogen level had the largest influence on the PI3-Akt signal pathway, with altogether 6 differentially expressed proteins being identified as the important signal molecules of that pathway, including five with known names and functions [14-3-3θ, OPN, GHR, IgG2a, and protein tyrosine kinase (PTK)] and one being unknown.
6、为进一步解析更年期综合征的发病机理，作者对鉴定到的磷酸化差异修饰蛋白进行了互作网络分析（PPI）。他们发现GLUT4和MAPK3为重要的节点蛋白质。
Figure 6. GLUT4 and MAPK3 as important node protein. PPI Analysis. There were two important node proteins among the three groups; glucose transporter 4 (GLUT4) and MAPK3. GLUT4 had the highest connectivity degree, interacting and associating with seven out of the differentially expressed proteins detected by MS.
小编心得：
本文是一篇典型的应用蛋白质组学技术分析揭示病理机制的文章。本文通过TMT蛋白质组分析揭示大鼠去卵巢后血浆蛋白质的变化情况，以及雌激素的治疗作用，发现了35种在更年期发挥重要作用的血浆差异表达蛋白质，以及其功能和参与的主要生物学通路。本研究这有助于更好地了解机体在围绝经期发生的变化的分子机制，并为围绝经期相关症状和疾病诊断生物标志物的挖掘和临床治疗方案的选择提供有价值的线索，也为后续的研究指明了方向。这篇文章显示了质谱技术在机理机制基础研究中的重要作用：差异表型整体性分析，全面系统性地解析潜在的分子机理。
文献来源：
Proteomics in plasma of ovariectomized rats and those exposed to estradiol valerate. J Steroid Biochem Mol Biol. 2017. (IF=4.561)
