基于3种非天然糖代谢标记的分泌蛋白质组分析性能对比  

Comparison of the performance of secretome analysis based on metabolic labeling by three unnatural sugars

作　　者：毛源 郑江南 封顺[1] 田瑞军 MAO Yuan;ZHENG Jiangnan;FENG Shun;TIAN Ruijun(School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China;Department of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen 518055, China)

机构地区：[1]西南交通大学生命科学与工程学院,四川成都610031 [2]南方科技大学理学院化学系,广东深圳518055

出　　处：《色谱》2021年第10期1086-1093,共8页Chinese Journal of Chromatography

基　　金：国家重点基础研究计划基金(2016YFA0501403,2016YFA0501404,2020YFE0202200);国家自然科学基金(31700088);广东省杰出青年基金(2019B151502050);深圳市科委创新基金(JCYJ20170412154126026).

摘　　要：分泌蛋白质是调控细胞间信号转导的重要生物大分子。由于分泌蛋白的丰度相比于胞内蛋白以及培养基添加剂更低,因此分泌蛋白的高通量鉴定是目前蛋白质组学界研究的热点和难点。目前,基于生物质谱的分泌蛋白质组学分析一般均需要从无血清的条件培养基中获得分泌蛋白质,再对其进行富集和分析。该流程操作步骤繁琐,易造成分泌蛋白质的损失和降解。本工作采用基于生物正交化学生物学技术实现对分泌蛋白质的高选择性标记和高效富集。通过结合点击化学技术,综合评估了分泌蛋白质分析中用于代谢标记的不同糖类似物。采用3种最常用的商品化糖类似物,N-叠氮乙酰甘露糖胺(ManNAz)、N-叠氮乙酰半乳糖胺(GalNAz)和N-叠氮乙酰葡萄糖胺(GlcNAz)分别对HeLa细胞进行代谢标记,之后通过炔基生物素探针对条件培养基中的分泌蛋白进行富集,结合质谱分析来对比3种糖类似物对分泌蛋白的标记效率。最后通过无标定量蛋白质组学分析,系统评估了3种糖类似物用于分泌蛋白质组分析的性能。结果表明,基于ManNAz的分泌蛋白标记方法鉴定到了282个分泌蛋白、224个细胞质膜蛋白以及846个N-糖基化位点;对分泌蛋白的富集效率分别较GalNAz和GlcNAz提高了130%和67.2%;对细胞质膜蛋白的富集效率较GalNAz和GlcNAz分别提高了273.3%和148.7%,体现出了明显的优势。本研究的实验结果为分泌蛋白高选择性富集和系统分析提供了有益的对比分析和新技术策略。Many secreted proteins,including cytokines,growth factors and hormones,are crucial in processes like intercellular signaling.Dynamic changes in secreted proteins usually reflect the growth and pathological state of the cells.Many drug targets are secretory proteins.The proteins are also important biomarkers.Conditioned cell culture media are important samples for secretory proteomic studies.Biomass spectrometry-based proteomic analysis enables the systematic study of secretory proteins.The main problem in analyzing secretory proteins in conditioned culture media is the low concentration of these proteins and the presence of serum,amino acids,and additives in culture media that may interfere with the protein analysis.Conventional secretory proteome analysis uses serum-free cell culture to reduce sample complexity,and typically involves protein concentration,purification,and desalting using ultrafiltration,dialysis,lyophilization,and trichloroacetic acid(TCA)or acetone precipitation,followed by enzymatic digestion and mass spectrometry analysis.This analytical process does not allow specific enrichment of secreted proteins.Thus,only a few secreted proteins can be identified.In addition,prolonged serum-free incubation of cells also tends to lead to unexpected changes in their activity status.A bioorthogonal-based enrichment approach can effectively avoid this problem.In recent years,unnatural sugars containing bio-orthologous groups,such as azide groups,have been used to metabolically label glycosylated proteins,enabling cellular imaging or selective enrichment of glycoproteins and their use for proteomic analysis.The strategy is a two-step process.First,azide-based sugar analogues are added to the cell culture medium and introduced to glycoproteins via the intracellular glycan biosynthesis pathway.Second,they are specifically covalently labeled with imaging probes or affinity probes via click chemistry.Since secreted proteins are usually glycoproteins,this glycolytic labeling has been used to label and enrich secre

关 键 词：液相色谱 串联质谱 分泌蛋白 点击化学 代谢标记 糖类似物 

分 类 号：O658[理学—分析化学]