Thermo的离子阱和蛋白质组学

1、2蛋白质组学概念及主要研究内容蛋白质组学技术基础蛋白质组学面临的挑战及其对策蛋白质鉴定的主要策略及技术流程样品前处理色谱/电泳分离质谱分析数据库检索3Proteome and Proteomics一个基因组、一个细胞或组织所表达的全部蛋白质一个基因组、一个细胞或组织所表达的全部蛋白质(PROTEOME = PROTEin + genOME )Proteomics 通过研究蛋白质组揭示基因组序列和细胞行为之间的关系通过研究蛋白质组揭示基因组序列和细胞行为之间的关系蛋白质组学是发现的科学，是系统生物学的重要组成部分蛋白质组学是发现的科学，是系统生物学的重要组成部分蛋白质的鉴定是蛋白质组学研究的基本

2、问题蛋白质的鉴定是蛋白质组学研究的基本问题4Protein IDQuantitationThroughputMSnPhosphorylationGlycosylationIntact Protein AnalysisProtein IsoformsBiomarkersDifferential ExpressionLabelsLabel-free5在蛋白质的水平上进行的蛋白质组学研究理想的蛋白质组学研究模式, 当前技术攻关的热点技术上存在困难, 近期难以实现通量化在多肽的水平上进行的蛋白质组学研究技术相对成熟,是当前蛋白质组学研究的主流信息不全6双向电泳和多维液相色谱技术生物质谱技术基因组计划的

3、完成生物信息学技术7Rodriguez-Ortega et al. Nat Biotechnol. 2006 Feb;24(2):191-7. 82DE 在蛋白水平上的分离在蛋白水平上的分离2DLC 在多肽水平上的分离在多肽水平上的分离9Look deeper1 mL1 L1 L1,000 L 或 1 L 起始体积MS，1 amol LOD血浆样品起始体积MS，1 fmol LODAlbuminIgGtissue leaking proteinsInterleukins cytokinesFibronectinComplement C110-3/mili10-6/micromol/L10-9/

4、nano10-12/pico10-15/femto10-18/atto 10-21/zepto10-24/yoctoInsulinPeptide drugs 51065103ng/mL, average M.W. = 5 kDa5100510-3510-6510-9unknownsProtein drugs10Science. 2006, Vol 312, 212-21711EICIFABAPCIESIMALDIAPPI12Ion Transfer TubeESI Needle+/- 5 kVTaylor ConeSolvent evaporation and Ion desolvation1

5、314Single quadrupole 单四极杆Triple quadrupole 三级四极杆Ion trap 离子阱TOF 飞行时间OrbitrapFTICRQ-TOFTOF-TOFQ-trapIon trap-TOFLTQ-OrbitrapLTQ-FT15CID/CAD 碰撞诱导裂解PSD 源后裂解SID/source fragmentation 源内裂解IRMPDECDETDPQD16Trypsin的作用位点在K、R的C-端肽键，专一性很强，水解蛋白产生的多肽（tryptic peptide）多在2-3 kDa，每个蛋白平均约产生50个肽段左右。Tryptic Peptide在ESI

6、positive mode下离子化效率高质谱在此质量范围能够准确测量，加上较多的肽段数及很强的专一性，保证了PMF策略鉴定蛋白质的可行性有效的MS/MS图谱一般来自大小在2-3 kDa 的多肽，且以R、K结尾的多肽裂解充分，有较强的规律性，保证了MS/MS策略的可行性。17PMF可以提供较高的序列覆盖率多采用2DE-MALDI-MS配置，适用于简单样品没有序列信息，可靠性差已经过时MS/MS提供序列信息，可靠性高多采用LC-ESI-MS/MS配置，适用于复杂样品存在采集时间限制问题广为接受18*measurements per hour2DE 500 - 10,000 protein spot

7、sAffinity Chromatography 2Ion exchange Chromatography 10protein levelGelfiltration 50 mg工业规模工业规模 2.5 l of plasma (K. Rose et al. in Proteomics 704) 但是，也不要上得太多但是，也不要上得太多!35Tandem MS36基本原则 先做一次全扫描（full MS)，记录个丰度最高的离子 然后依次对这X个离子进行CID, 做MS/MS(full MS2) 然后再做全扫描, 下一个data dependent MS/MS开始Dynamic exclusion

8、 某个离子在做了一定次数（比如2次）的MS/MS后，将在一定时间内（比如3min）不再作为侯选离子。37自动LC/MS/MS肽序列测定38+多肽的裂解39碎片离子的命名40 Residue name (A-Z) monoisotopic massaverage massA71.03711 71.0788B114.53493114.59625C103.00919103.1388D115.02694115.0886E129.04259129.1155F147.06841147.1766G57.02146 57.0520H137.05891137.1412I113.08406113.1595J0.0

9、0.0K128.09496128.1742L113.08406113.1595M131.04049131.1925N114.04293114.1039O0.00.0P97.05276 97.1167Q128.05858128.1308R156.10111156.1876S87.0320387.0782T101.04768101.1051U150.95364150.034V99.06841 99.1326W186.07931186.2133X111.0111.0Y163.06333163.1760Z128.55059128.62315氨基酸残基质量41在低能CID条件下，tryptic pept

10、ides主要产生以y离子和b离子为主的碎片离子此外还经常可以看到y离子、b离子中性丢失NH3或H2O的峰脯氨酸（P）残基N-端侧的肽键特别脆弱，因此含P的多肽MS/MS图谱一般会有一个脯氨酸N-端侧的肽键断裂产生的特别强y离子。而脯氨酸（P）残基C-端侧的肽键一般不断，因此相应的碎片离子基本不出现如果肽段中R数小于其所带质子（H+），肽段将以charge directed 方式裂解，碎片离子丰富；反之，则以charge remote方式裂解，碎片离子较少。在charge remote方式下D、E等酸性氨基酸残基C-端肽键易断糖肽上的寡糖基、磷酸肽上的磷酸基在CID条件下更容易脱落，因此该类多肽

11、的MS/MS图谱的base peak一般为母离子脱去修饰基团后产生的4243#9296-9296 RT:104.79-104.79 NL: 2.89E240060080010001200140016001800m/z05101520253035404550556065707580859095100Relative Abundancey y8+1925.5b b8+1921.4y y10+11125.7y y13+11468.4y y6+1720.4b b12+11325.9y y11+11240.7y y5+1623.5b b4+1411.2y y3+1411.2b b9+11009.4y y

12、14+11635.7b b10+11122.4y*y*14+2809.5yoyo4+1506.3LPSIS*DLDS*IFGPVLSPK=974.5M+2H - H3PO42+(2047/298)-Prominent loss of phosphoric acid from M+2H2+ ion4445首先确定母离子的质量在数据库中按一定误差检索符合条件的肽段将MS/MS中观察到的子离子质量与数据库中多肽的子离子理论值比较找到最匹配的SequestMascot46 PERFORMANCEExceptional coverageHigh sensitivity MSnUltimate Perfo

13、rmanceRedefining Accurate Mass 47Blackler, A. R.etc Anal. Chem.; 2006; 78(4); 1337-1344. Viveka Mayya, Karim Rezaul, Yu-Sheng Cong, and David Han. Molecular & Cellular Proteomics 4:214223, 2005.More protein identifications, low false positives48 Ion Trap based Fourier Transform ICR Mass Spectrom

14、eter FTICR (FTMS) at LC timescale Accurate Mass High Sensitivity Ultra-high Resolution Above All - Simple to Operate49 Advanced LC-MS Proteomics Metabolomics Pharmaceutical Drug Discovery Small Molecule and Structure Elucidation Ultra-trace Level Analysis505152Sample PreparationBiomarker ResearchDat

15、aInterpretation& StorageSample AnalysisRoboticsCentrifugesConcentratorsLiquid Chromatography/Mass SpectrometryProtein IdentificationProtein Digest Sample HandlingData Analysis Peptide SeparationData StorageLabInformationManagementSystem Microplate ReadersSoftwareXcaliburProteinFractionationCells

16、 or TissueCells or TissueBiomarkerIdentifiedFreezersCompany Confidential54Its a jungle out thereSILACAQUAcICATiTRAQIsotopeLabelsIsotope-CodedTagsICAT and iTRAQ are registered trademarks of Applera CorporationAQUA is a registered trademark of Harvard University55 Isotope ICAT, cICAT, iTRAQTags Global

17、, Complex, Expensive, Non-parallel workflowLimited practical successIsotopeSILAC (amino acids)Labels Metabolic Labeling - Enriched Media (15N)Global, InexpensiveLimited to cells grown in cultureMethod of Choice for Biological Hypothesis TestingAQUA Labeled internal standard for every protein of inte

18、restBest sensitivity, accuracy and precisionMethod of Choice for Biomarker ValidationLabel-MS Peak Area RatiosFreeGlobal, “Free”Method of Choice for Biomarker Discovery 56Validation Rather than continue to look for all proteins,develop a method to look only at a list of targeted proteins. Immunoassa

19、y? Multiplexed MS analysis of Labeled Peptides?Structural Characterization Exact protein isoform Top Down analysis57For every protein, there are a number of peptides which can be used to specifically identify that protein.These are called “Proteotypic Peptides”In a shotgun sequencing experiment, bas

20、ed on LC/MS data,these are expected to be 1) Tryptic peptides2) Without commonly modified amino acids (M, S, T, Y)3) Good ionization efficiency for MS/MS analysis- Reudi Aebersold58Identify Proteotypic Peptides for proteins of interestOrder/synthesize isotopically enriched internal standards for the

21、se peptidesSpike a known amount of the labeled peptide(s) into sampleDigest samplePerform LC-MS/MS, following both the labeled internal standard(s) and non-labeled peptide(s) of interest (LC-SRM)Calculate peak area ratio for standard and unlabeled peptide 59qMass range m/z 30-3000q 300 H-SRM transit

22、ionsqMulti-residue screening methodsFAIMS enabled (food residues)Intact protein analysisdetermines exact protein isoform6163Nuclear membraneChromatin fiberNuclear poreNucleosomesDNAChromatin fiber(30 nm dia.)H1CoreHistonesH3H4H2BH2ANuclear membraneChromatin fiberNuclear poreNucleosomesDNAChromatin fiber(30 nm dia.)H1CoreHistonesH3H4H2BH2Aadapted from Mathews et al.,