干旱区生态环境知识资源中心(Arid): 尿液多肽组的高效富集与快速鉴定方法优化

Arid

	尿液多肽组的高效富集与快速鉴定方法优化
其他题名	Optimization of efficient enrichment and rapid identification of urine endogenous peptidomes
	王丹阳 1; 王一豪 2; 兰秋艳 2; 李衍常 2; 徐平 2; 韦锦斌 3
来源期刊	军事医学
ISSN	1674-9960
出版年	2018
卷号	42 期号:12 页码:916-923
中文摘要	目的对尿液多肽组提取方法进行优化,简化尿液多肽组样品的制备流程,提高多肽的富集效率与鉴定数量,为潜在的临床生物标志物筛选提供技术支持。方法将3 ml尿液样品经超滤管[截留量为1010~3相对分子质量(Mr)]离心后得到流穿液,通过脱盐层析柱(PD-10)除去流穿液中的盐及尿黄素等小分子成分,得到多肽组样品,然后利用纳升高效液相色谱-高分辨串联质谱(nano-UPLC-MS/MS)进行检测。为提高多肽组的测序深度,可提高尿液样品起始量(如10~40 ml),经过超滤管离心后,利用反相色谱柱(Oasis HLB固相萃取柱)进行富集浓缩,经PD-10脱盐和StageTip分离后,得到可用于质谱检测的尿液多肽组样品。研究中处理尿液样品总时间控制在4~6 h,脱盐过程简单,在10 ml尿液样品中最多鉴定到归属于261种蛋白的1810条肽段。结果使用1010~3 Mr截留量超滤管所得多肽样品鉴定到的肽段数量是3*10~3 Mr截留量超滤管的1.5倍,同时,前者可有效缩短样品处理时间;使用PD-10层析柱能有效地除去尿液样品中的尿黄素等小分子物质,提高了质谱检测效率;质谱数据分析选用pFind开放式搜库模式,与MaxQuant相比能显著提高多肽的鉴定率,且搜库耗时显著缩短;利用定量蛋白质组学策略,评估了该方法对多肽的回收效率,其平均回收效率>50%,最高>90%。结论通过对尿液多肽组富集与检测的各个环节的测试与优化,显著降低了尿液多肽组的检测时间和成本,简化了实验操作流程,所需尿液样品体积更小,同时可实现较高规模的多肽组测序覆盖度,为筛选潜在的疾病生物标志物提供了技术支撑。
英文摘要	Objective To optimize the extraction method of urine endogenous peptidomes,simplify the sample preparation process and improve the enrichment efficiency so as to provide technical support for finding clinically biomarkers from urine.Methods The urine peptidome was obtained via centrifugation through the ultrafiltration tube of 1010~3 Mr.The small molecule components such as salt and urine flavin were removed directly through the PD-10 column.Finally,the resulting peptides were analyzed through nano-LC-MS/MS.In order to improve peptidome coverage,urine samples with larger volumes,such as 10-40 ml,were first centrifugated through the 1010~3 Mr ultrafiltration tube and then through the Oasis HLB solid phase extraction column for sample concentration.After being desalted by PD-10 column,the resulting peptides were separated by StageTip column.The total time of sample processing was confined to 4-6 h.The desalting process was simple,and 1810 peptides belonging to 261 proteins were identified in 10 ml urine samples.Results The number of peptides identified using the 1010~3 Mr ultrafiltration tube was 1.5 times that of the 310~3 Mr ultrafitration tube.Additionally,the sample processing time could be shortened considerably.The use of the PD-10 column could effectively remove the salt composition and small molecules such as urine flavin.For the MS data analysis,the software pFind could improve the identification quantity compared with MaxQuant,and much less time was taken.Using the strategy of quantitative proteomics,the average recovery efficiency of peptides by this enriched method was more than 50% and up to 90%.Conclusion This experimental method can significantly save the time and cost of urine peptidome detection.Moreover,the steps are simpler,and require fewer urine samples,which can provide methodological support for the future clinical applications.
中文关键词	尿液 ; 多肽组 ; PD-10脱盐层析柱 ; 纳升液相色谱-高分辨串联质谱
英文关键词	pFind urine peptidomics PD-10 desalination chromatography column nano-UPLC-MS/MS pFind
语种	中文
国家	中国
收录类别	CSCD
WOS类目	MEDICINE GENERAL INTERNAL
WOS研究方向	General & Internal Medicine
CSCD记录号	CSCD:6482092
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/237886
作者单位	1.广西医科大学药学院;;军事科学院军事医学研究院生命组学研究所,国家蛋白质科学中心(北京), ;;蛋白质组学国家重点实验室, 南宁;;, ;;北京 530021;;102206; 2.军事科学院军事医学研究院生命组学研究所,国家蛋白质科学中心(北京), 蛋白质组学国家重点实验室, 北京 102206, 中国; 3.广西医科大学药学院, 南宁, 广西 530021, 中国
推荐引用方式 GB/T 7714	王丹阳,王一豪,兰秋艳,等. 尿液多肽组的高效富集与快速鉴定方法优化[J],2018,42(12):916-923.
APA	王丹阳,王一豪,兰秋艳,李衍常,徐平,&韦锦斌.(2018).尿液多肽组的高效富集与快速鉴定方法优化.军事医学,42(12),916-923.
MLA	王丹阳,et al."尿液多肽组的高效富集与快速鉴定方法优化".军事医学 42.12(2018):916-923.