非标记液质联用蛋白组分析中国实验小型猪冠心病模型的方法研究

目的: 针对中国实验小型猪冠心病模型,建立非标记液相色谱/质谱联用血清蛋白组学研究方法。 方法: 研究中分别设立健康对照组、模型组、阳性药物治疗组及中药治疗组,分析过程中,样品未进行高丰度蛋白去除,利用BCA法测定蛋白浓度后,直接使用-20 ℃冷丙酮冰上沉淀,4 ℃ 放置过夜,离心,弃去溶液,对蛋白沉淀,分两步加入Trypsin进行酶解处理,加酸终止后,离心,获取上清液进行分析。测定方法采用160nl Trap柱进行蛋白富集,分析柱采用Agilent Chip(C₁₈,150 mm×75 μm),为获得较高的蛋白覆盖率,方法进行了如下优化:测定梯度优化在97%水相-77%水相区间;线性梯度运行时间为125 min;Data dependent 参数优化;2次重复进样等。测定结果利用Agilent Spectral mill进行搜索,部分蛋白利用Mascot进行了验证搜索。 结果: 最终在健康对照组获得特异性蛋白512个(unique protein),重叠蛋白47个(overlapped)。以获得的健康组特异性蛋白为参照,对4组来源样品进行了PCA分析,将上述各组进行分类,其中模型组与治疗组得到了明显的区分,中药治疗组与阳性药组有着相似的变化轨迹,但与健康组区分明显,这说明实验中大部分冠心病的损伤存在很大程度的不可逆性,药物治疗仅对其中部分蛋白有着转归趋势,例如对其中纤维蛋白原,有着明显的下调趋势。研究还针对主要的蛋白进行了功能分类,相对识别了模型组与健康组、模型组与治疗组;治疗组与健康组之间的蛋白上调及下调趋势,其功能主要集中在脂质代谢相关的蛋白,如Apoliprotein,HAS,及C 反应蛋白。 结论: 方法证明了利用非标记蛋白鉴定技术,可以较好地评价出动物模型的变化,同时可以在一定程度上反映出治疗的转归方向。

Objective: A label free liquid chromatography/mass spectrometry(LC/MS)based serum proteomics method is developed on China mini pig model with experimental coronary heart disease in current work. Methods: Global proteomics analysis had been executed on serum samples collected from healthy control group,model group,positive drug therapy,and Traditional Chinese Medicine(TCM)treatment group.The total contents of protein in serum samples without wiping off high abundance protein were determined by Bicinchoninic acid Protein Assays(BCA).Serum samples were precipitated with -20 ℃ cold acetone on ice,placing overnight at 4 ℃,discarding the solution after centrifuging.The protein pellets were digested in two steps with trypsin.Reactions were terminated by acid and the supernatants were applied to analyzing after centrifuged.The method was completed on a 160nl trap column for enrichment and an Agilent Chip(C₁₈,150 mm×75 μm)for separation.In order to obtain high protein coverage,test conditions were optimized as follows: gradient window was 97%to 77%hydrous mobile phase,run time for linear gradients was 125 min,parameters optimized on data dependent,and replicated sample tests,etc.. Results: Results were searched with Agilent Spectral mill and partial validated with Mascot with relative rigid parameters.512 proteins with unique peptides and 47 overlapped were found on healthy group in final results.Principal Component Analysis(PCA)was used to clarify the data from four experimental groups with the proteins results from healthy as reference.The results showed distinct classification between model and drug treatment group,while the similar trajectories were obtained between positive drug and TCM group.All results also illustrate the fact in our experiments that the injuries from coronary heart disease are irreversible changes to a large extent,while drug therapy is only working on prognosis tendency for partial proteins,such as fibrin,which has clearly down regulated trend.Current studies also aim at the major functional classification of proteins between model and health groups,as well as model and treatment groups.The up and down regulated trends in proteins between treatment and healthy group reveal the major functional changes on heart disease are focusing on lipid metabolism-related proteins,such as Apoliprotein,HAS,and c-reactive protein. Conclusion: All proofs from current method give us the evidence that the label free LC/MS can be a useful tool to evaluate the protein changes across animal models,and also reflect the partial prognosis outcome of the treatment.