两种不同分离方法的唾液多肽组分析结果比较  

Comparison of Two Different Separation Methods for Analysis of Salivary Peptidome

作　　者：孔祥怡[1] 杜建时[1] 徐金玲 李水明 王勇 赵晴[3] KONG Xiang-Yi;DU Jian-Shi;XU Jin-Ling;LI Shui-Ming;WANG Yong;ZHAO Qing(Lymphatic and Vascular Surgery Department, Xinmin Branch of China Japan Union Hospital,Jilin University, Changchun 130033, China;College of Life Science and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen University, Shenzhen 518060, China;Neurology Department, Nanhu Branch of China Japan Union Hospital, Jilin University, Changchun 130033, China)

机构地区：[1]吉林大学中日联谊医院新民淋巴血管外科,长春130033 [2]深圳大学生命与海洋科学学院,深圳市海洋生物资源与生态环境重点实验室,深圳518060 [3]吉林大学中日联谊医院南湖神经内科,长春130033

出　　处：《分析化学》2019年第11期1816-1822,共7页Chinese Journal of Analytical Chemistry

基　　金：吉林省科技发展计划(No.2018041607FG);广东省重点领域研发计划2018-2019年度“脑科学与类脑研究”重大科技专项项目(No.2018B030336001);深圳市科技项目(No.JCYJ20170818142154551)资助

摘　　要：唾液多肽经Zip-Tip C 18固相萃取和氧化石墨烯-磷酸镧纳米复合材料(LaGM)两种方法分离后,利用高分辨串联飞行时间质谱进行多肽鉴定,将重复两次的结果合并,比较两种方法的异同。利用Zip-Tip C 18方法共检测到归属于38种蛋白质的545条序列特异肽段,而LaGM方法检测到了来源于44种蛋白质的359条序列特异肽段,其中二者重合的有116条肽段,归属于21种蛋白质。两种方法所得的肽段分布特征和优势肽段构成存在明显差异,Zip-Tip C 18更多富集Submaxillary gland androgen-regulated protein 3B、Statherin和Salivary acidic proline-rich phosphoprotein 1/2等蛋白质的肽段,而利用LaGM方法可检测更多的来源于Histatin-1、Histatin-3和Protein S100-A8的肽段,考虑翻译后修饰,结论一致。以上结果表明,这两种方法对多肽的富集有一定偏好性,即多肽分离过程有可能导致肽段的特异性丢失。进一步分析发现,丢失肽段可能是检测到肽段的序列相关肽段、修饰相关肽段、所归属蛋白质的其它肽段或其它蛋白质的肽段,但序列相关肽段的几率更大。本研究表明,不宜用单一分离方法的结果代表整个多肽组,LaGM方法虽然检出肽段数目较少,但可以获取更多的降解蛋白质的信息。Zip-Tip C 18 solid phase extraction and oxide graphene-lanthanum phosphate nano composite (LaGM) were used to separate saliva peptides respectively. The peptides were identified by high-resolution tandem time-of-flight mass spectrometry (TOF-MS). To reduce accidental errors, the MS analysis repeated once, and the results of two replicates were combined and then were compared at the peptide and protein levels respectively. A total of 545 sequence-specific peptides belonging to 38 proteins were detected by Zip-Tip C 18 method, and 359 different peptides coming from 44 proteins were detected by LaGM method, and the two were coincident with 116 peptides (19 proteins). Furthermore, it was found that the peptide distribution characteristics and the dominant peptide composition obtained by the two methods were significantly different. More peptides of Submaxillary gland androgen-regulated protein 3B, Statherin and Salivary acidic proline-rich phosphoprotein 1/2 were enriched by Zip-Tip C 18;more peptides of Histatin-1, Hisatin-3 and Protein S100-A8 were detected by LaGM method, and the conclusions were consistent after post-translational modification were included. This study showed that these two methods had a preference for the enrichment of peptides, e.g. the separation process may lead to the loss of some peptides, the possible missing peptides may be the sequence-related peptides, modification-related peptides, protein-related peptides or peptides coming from some other proteins, fortunately, the potential missing peptides were more likely to be related with the peptides that had been detected. This study suggested that it may be not appropriate to use the results of a single separation method to represent the entire peptidome, and although the LaGM method detected fewer peptides, it was more conducive to enrichment of low-abundance peptides, by which more information about degrading proteins could be obtained.

关 键 词：多肽组 Zip-Tip C 18 氧化石墨烯-磷酸镧纳米复合材料 唾液 高分辨串联质谱 

分 类 号：O652.6[理学—分析化学] O629.7[理学—化学]