家蚕微孢子虫总蛋白两条大分子量主带的LC-MS/MS分析

LC-MS/MS Analysis of Two Main Bands with High Molecular Weight in Total Proteins of Nosema bombycis

为了深入认识家蚕微孢子虫高分子量蛋白的主要组成,我们采用LC-MS/MS技术鉴定分析玻璃珠破碎法提取的家蚕微孢子虫总蛋白中85kD以上两条主要蛋白带。我们从H1带(位于150～200kD之间)中成功鉴定出16个蛋白,包括极管蛋白3(PTP3)、极管蛋白2(PTP2)、真核蛋白翻译起始因子(eIF2C2)、延伸因子(EF1-alpha、EF2)、肌动蛋白(actin)等。这暗示两种结构蛋白PTP3与PTP2以及蛋白翻译延伸因子(EF1-alpha、EF2)与肌动蛋白(actin)可能各形成一个复合体。并且用SDS或β-巯基乙醇处理时,这两个复合物是稳定的,表明这些复合物的相互作用力不仅仅是通过二硫键。H2带(85～100kD)鉴定出主要集中在水解酶、蛋白合成与折叠、细胞结构、运动四个方面的20多个的蛋白。从两条蛋白带鉴定出PTP3与PTP2、三磷酸腺苷酶(ATPase)或过渡期内质网三磷酸腺苷酶(ATPase-TER94)、蛋白翻译因子(eIF2C、EF1-alpha、EF2)、HSP70、Actin、M1家族氨肽酶1(M1family aminopeptidase)6类蛋白。基于蛋白分子质量分析,暗示在家蚕微孢子虫中,这6类蛋白都可能是以复合体形式呈现的。研究结果丰富家蚕微孢子虫高分子量蛋白的数据,同时,为研究非二硫键间的蛋白互作提供了有价值的研究线索。

In the post-genome era,the combination data with mass spectrometry （MS） provide an effective method for protein identification, and MS has been developed into a powerful tool for proteomics. Now, of Nosema bombycis proteins, only a few have been identified, inclu- ding some spore wall proteins and polar tube proteins. In order to obtain more information of N. bombycis proteins with big molecular weight,we used the technology of proteomics, LC- MS/MS, in this study to identify and analyze two abundant protein bands of glass bead-ex- tracted total proteins of N. bombycis. We successfully identified a total of 16 proteins in H1 （130--170 kDa） band, including polar tube protein 3 （PTP3）, polar tube protein 2 （PTP2）, eukaryotic translation initiation factor 2C 2 （eIF2C 2）, elongation factor 1-alpha （EFI-al- pha）, elongation factor 2 （EF2）, actin and some others, thus suggesting that the two struc tural proteins （PTP2 and PTP3） could form a complex, and the translational related pro- teins, eukaryotic translation elongation factor 1-alpha（EFl-alpha）, elongation factor 2 （EF2） and actin could form a big complex too. Treated with SDS or t3-Mercatoethanol, these two complexes were stable, which suggested that their ligation is not by the disulfide bond alone. Over 20 proteins were identified in the H2 band with a molecular weight size ranging from 85 kDa to 100 kDa, such as adenosine triphosphatas （ATPase） ,transitional endoplasmic reticu- lum ATPase TER94, M1 family aminopeptidase 1, transitional endoplasmic, PTP3, PTP2, elF2C 2,EF2,EF1 alpha and so on. According to functional analysis, these proteins mainly included the following four aspects, hydrolytic enzymes,protein synthesis and folding,cellu lar structure and movement. Our research results provide more basic data for proteome and some clues for further research of protein interaction in microsporidia.