利用iTRAQ技术联合2D LC-MS研究不同加工工艺鹿茸的差异蛋白质组学

Comparative proteomics study of different processing technology for pilose antler using iTRAQ technology coupled with 2D LC-MS

利用i TRAQ同位素标记法结合2D LC-MS/MS法对不同加工工艺鹿茸(鲜品、煮炸、直接冻干、匀浆、冻干加保护剂、冻干未加保护剂、冻干加保护剂40℃,10天)进行比较蛋白质组研究。经质谱共分析共得到蛋白质1 015种。利用Protein Pilot软件(Version 4.5)进行比较分析,差异蛋白(阈值在>1.50或<0.60范围,P≤0.05)蛋白为87种。变化最大的是煮炸鹿茸组,其差异蛋白质(P≤0.05)相对于鹿茸鲜品,上调蛋白质有24种,下调蛋白质有33种。7种差异极显著(P≤0.001)下调蛋白多具有参与钙离子结合及ATP结合等功能。其他组差异蛋白质种类没有明显变化,与通过层次聚类分析的结果相同。冻干加保护剂(海藻糖)工艺可提高差异极显著蛋白质包括II型、XII型胶原蛋白质(P≤0.001)的相对含量,并且使得功能显著蛋白质与鲜品鹿茸比较,含量相对增加。这些蛋白具有活化血小板、维护精原细胞、使肿瘤细胞表达失调等生理功能。通过对差异蛋白质的层次聚类(hierarchical clustering)及GO(gene ontology)注释分析,表明不同加工工艺鹿茸与鲜品鹿茸的差异蛋白质在分子功能、生物过程及细胞成分中,具有广泛的生物学功能覆盖范围,其中分子功能主要体现在结合功能(52.7%)、催化活性功能(25.3%)、结构分子活性及转运功能(6.6%)。

This study was designed to use iTRAQ technology coupled with 2D LC-MS/MS to study the comparative proteomics of different processing technology for pilose antler. 1015 proteins were identified with 2D LC combined with MOLDI TOF/TOF mass spectrometry. Comparative analysis with Protein Pilot （Version 4.5） revealed that 87 proteins were changed （P≤0.05, the ratio of 〉 1.50 or 〈0.60 as the threshold selection of difference proteins）, of which 24 were up regulated and 33 were down regulated in the traditional frying process （TFP） compared with the fresh pilose antler （P≤0.05）. 7 significant different proteins （P≤0.001）, most of these significantly changed proteins were found to be involved in calcium ion binding and ATP binding associated with human healthy, Freeze drying with protective agent （FDP） （Trehalose） can improve the content of significantly different proteins （P≤ 0.001） including Collagen alpha- 1 （XII） chain （COL 12A 1） and Collagen alpha- 1 （II） chain （COL2A1）. The significant function involves in platelets activating, maintenance of spermatogonium, and disorder expression in tumor cells. The functional annotation by Hierarchical clustering and GO （gene ontology） showed that the main molecule functions of the proteins significantly changed in these processes were involved in binding （52.7%）, catalytic （25.3%）, structural molecule and transporter （6.6%）.