血浆血清体液蛋白质组学(改进版柳满然)技术方案.ppt

* 常用串联质谱检索工具 SEQUEST: F/sequest PepFrag: P ProteinProspector: P Mascot: * ESI-MS/MS MAIDI-TOF 图像分析 确定差异点 差异蛋白质质谱分析 数据处理与生物信息学分析 数据搜寻蛋白质鉴定 条件A:样品 条件B:样品 基于2DE的差异蛋白质组分析技术流程 IEF:利用等电点差异分离Pr SDS:利用分子量差异分离Pr * Provides a hard-copy record of separation Allows facile quantitation Separation of up to 3000 different proteins Highly reproducible Gives info on Mw, pI and post-trans modifications Inexpensive Limited pI range (4-8) Proteins 150 kD not seen in 2D gels Difficult to see membrane proteins (30% of all proteins) Only detects high abundance proteins (top 30% typically) Time consuming 30% of spots with multiple proteins from pI 4-7 2DE:Advantages Disadvantages * 传统的基于2DE的差异蛋白质组学分析技术 优点： ①技术路线成熟 ②重现性和直观性好 ③费用较低 ④展示差异蛋白质的PI和Mw 缺点： ①蛋白质共迁移(comigration)问题，不适合于很复杂的样品 ②线性动态范围较窄 ③强疏水性，强硷性，分子量过大(?150kD）的 蛋白质受到限制 * Proteins quantification Quantitative analysis SCX RP 2D chromatographic separation of peptides 数据处理与 生物信息学分析 ICAT label cysteines heavy light Combine trypsinize heavy light 基于Shotgun差异蛋白质组分析技术流程 iTRAQ-LC-MS/MS * Shotgun Protein Identification Protein mixture Protein digests SCX column fractionation Reverse column separation Auto MS/MS detection Tandem MS spectra BioWorks data base search Results * Alleviate some limitations of the 2-DE/MS method Generally high throughput Protein identification and quantification is straightforward process is simple Can be used to ID post-trans. modifications Requires more handling for sample isotopic labeling Low reproducibility Requires high level expertise Showed biases for high Mr proteins and against certain types and classes of proteins Advantages Disadvantages Shotgun:Advantages Disadvantages * Sensitivity and dynamic range of protein analysis methods Sensitivity Dynamic Range (Orders of Magnitude) Bio-Safe coomassie G-250 stain 5-10ng 2-3 Coomassie Blue R-250 stain 10-25ng 2-3 Silver stain kit 0.5-1ng 1-2 SYPRO Ruby protein gel stain 0.5-1ng 3-4 MALDI-TOF/TOF-MS 0.5-1pg 3-4 ESI-MS/MS (ion trap) 0.5