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

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 secreted proteins.N-Azidoacetylgalactosamine(GalNAz),N-azidoacetylglucosamine(GlcNAz),and N-azidoacetylmannosamine(ManNAz)are classical azide-based sugar analogues.Their effects on cytoplasmic membrane proteins have been compared.However,only ManNAz has been used for metabolic labeling of secreted proteins.No other glyco-analogues that label secreted proteins have been reported.Here,the bio-orthogonal chemical biology technology achieved highly selective labeling and enriched secreted proteins.In combination with click chemistry,different sugar analogues were evaluated for metabolic labeling of secreted proteins.HeLa cells were metabolically labeled by ManNAz,GalNAz,and GlcNAz(the three most commonly used commercial sugar analogues).These glycolytic markers can selectively label specific types of glycosylation.For example,ManNAz,an analogue of the biosynthetic precursor of sialic acid,N-acetylmannosamine(ManNAc),can label sialylated N-or O-glycoproteins.GalNAz,an analogue of N-acetylgalactosamine(GalNAc),can replace GalNAc as a core residue of mucin-type O-glycans and thus label O-glycoproteins.In addition,the intracellular metabolic intermediate of GalNAz(pyrophosphate)UDP-GalNAz can be interconverted with UDP-GlcNAz catalyzed by UDP-galactose-4-differential isomerase(GALE)and thus can also label N-glycoproteins and O-GlcNAc glycoproteins instead of GlcNAc.The GlcNAz analogue is commonly used to label nuclear and cytoplasmic glycoproteins withβ-O-GlcNAc residues,but can also label N-glycoproteins with mucin-type O-glycoproteins by converting GALE to GalNAz,followed by enrichment using a biotin-alkynyl probe.Label-free quantitative proteomic analysis was performed to evaluate their labeling efficiency.ManNAz-based secretory protein labeling identified 282 secretory proteins,224 plasma membrane proteins,and 846 N-glycosites.Compared with GalNAz and GlcNAz,the enrichment of secreted proteins was increased 130%and 67.2%,respectively,and the enrichment of plasma membrane proteins was increased 273.3%and 148.7%,respectively.This study provides a useful comparative analysis and new strategies for highly selective enrichment and systematic secretome analysis.