Thermo的离子阱和蛋白质组学ppt课件

蛋白质组学与生物质谱技术,主要内容,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,Proteome and Proteomics,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问题,Proteomicsn: Proven Thermo Solutions,Protein IDQuantitationThroughput,MSnPhosphorylationGlycosylation,Intact Protein AnalysisProtein IsoformsBiomarkers,Differential ExpressionLabelsLabel-free,Bottum-up and Top-down,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,蛋白质组学的基础和前提,双向电泳和多维液相色谱技术 生物质谱技术 基因组计划的完成 生物信息学技术,Promise of proteomics,Rodriguez-Ortega et al. Nat Biotechnol. 2006 Feb;24(2):191-7.,蛋白质组的复杂性 vs 蛋白质组学中的分离策略,2DE 在蛋白水平上的分离 2DLC 在多肽水平上的分离,Challenges in proteomics,Look deeper,1 mL,1 L,1 L,1,000 L 或 1 L 起始体积 MS，1 amol LOD,血浆样品起始体积 MS，1 fmol LOD,Albumin,IgG,tissue leaking proteins,Interleukinscytokines,Fibronectin,Complement C1,10-3/mili,10-6/micro,mol/L,10-9/nano,10-12/pico,10-15/femto,10-18/atto,10-21/zepto,10-24/yocto,InsulinPeptide drugs,5106,5103,ng/mL, average M.W. = 5 kDa,5100,510-3,510-6,510-9,unknowns,Protein drugs,质谱,Science. 2006, Vol 312, 212-217,离子源,EI CI FAB APCI ESI MALDI APPI,Ion Transfer Tube,ESI Needle,+/- 5 kV,Taylor Cone,Solvent evaporation and Ion desolvation,ESI: Electrospray ionization,MALDI：matrix assisted laser desorption ionization,质量分析器,Single quadrupole 单四极杆 Triple quadrupole 三级四极杆 Ion trap 离子阱 TOF 飞行时间 Orbitrap FTICR Q-TOF TOF-TOF Q-trap Ion trap-TOF LTQ-Orbitrap LTQ-FT ,裂解方式,CID/CAD 碰撞诱导裂解 PSD 源后裂解 SID/source fragmentation 源内裂解 IRMPD ECD ETD PQD ,Why trypsin?,Trypsin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 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。,PMF vs. MS/MS searching,PMF 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,Separation power Peak capacities,*measurements per hour,2DE 500 - 10,000 protein spots Affinity Chromatography 2 Ion exchange Chromatography 10 protein level Gelfiltration 10 Reversed Phase Chromatography 50 Ion Exchange Chromatography 25 Reversed Phase Chromatography 100 peptide level 2DLC (IEX/RPC) 2,500 Linear IT MSn 18,000 mph*,Why 2D LC/MS?,可鉴定具有极端pI、疏水性、分子量的蛋白质 适合膜蛋白 重复性好 容易自动化 上样量大 高灵敏度（溶液酶切） 可根据样品灵活配置（但最后一维一般为RP）,Then why SCX/RP/MS?,SCX和RP的分离原理是互补的 SCX按荷电情况，PR按疏水性 流动相是兼容的 pH3，ACN/water/salt SCX流动相中的盐可以在RP时有效去除 Tryptic peptides 在SCX柱上可以有效保留 Tryptic peptides在SCX条件下是稳定的,Copyright 2004 by the National Academy of Sciences,Beausoleil, Sean A. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 12130-12135,Fig. 1. Scheme for phosphopeptide enrichment by SCX chromatography,Gel,Gel spot,Proteolytic peptides,Mixture of Proteins,Digestion,Proteolytic peptides,Digestion,LC,Protein Identification Workflows,Peak Finding,Peak list,Search of a Sequence Collection,Identified and Proteins,Protein Candidates,Significance Testing,Protein Function?,Sequence Similarity Search,样品的前处理,Very important process (VIP)! 样品的损失 吸附 样品的污染 角蛋白 酶切不充分 溶液酶切和胶内酶切 ,How many peptides have we lost?,The spectrum of fraction 108 after deletion of ACN by N2 flow.,The spectrum of the ACN elute of the tube of fraction 108 after deletion of ACN by N2 flow.,The spectrum of fraction 108 after deletion of ACN by N2 flow and ZipTip erichment.,回收率和 操作步骤多少的关系 Keep it simple!,Total no. of steps,Overall % recovery,100 % recovery/step,90 %,80 %,70 %,60 %,50 %,MudPIT Multi dimensional Protein Identification Technology,在Yates 发表关于MudPIT 的文章的许多年前就有人发表关于2D LC的文章。但是Yates第一个将2D LC用于蛋白质组学并引起学术界的广泛关注。此后，各种各样的类似2D LC方法应运而生，其中有一些方法得到广泛接受和应用。,2DLC 2-dimensional liquid chromatography,MDLC Multi Dimensional Liquid Chromatography,RPC,IEX,RPC trap,RPC trap,on-line elution by salt plugs to trap column,IEX,off-line gradient elution with “classical” fraction collection,RPC,2D LC的三种配置形式,IEX,RPC,in-line MudPIT,MudPIT,SCX,RPC,优点 构造简单 SCX 和 RPC 填料在同一个柱子里 自己装柱，成本较底,缺点 方法和柱子没有商业化 SCX和RPC都不在最佳状态 必须得用挥发性缓冲体系 由于需要很长的平衡时间，因而费时,on-line 2D-LC,优点 全自动 样品体积可根据后续RPC调整 在线脱盐、浓缩 自由选择缓冲盐,缺点 SCX峰容量有限 ACN浓度在IEX和RPC得一致 两相分离都没能在最佳状态下进行,off-line SCX with fraction collection,优点 两维分离都有可能在最佳状态下进行，得到最好分辨率和峰容量 可自由选择缓冲体系 样品体积可根据后续RPC调整 速度快（只需一个SCX操作） SCX馏份可留待以后分析,缺点 自动化程度低,off-line SCX 的典型结果,Column: Mini S PC 3.2/30, NPR, 3 m Eluent A: 0.065% TFA in 30%ACN Eluent B: 500 mM KCl in Eluent A Typical peak width: ca. 10 s,The secret behind sensitivity Reducing column dimensions,75 m I.D. columns and 200 nl/min are the standard today,75 m I.D. columns and 200 nl/min are the standard today,off-line 2D LC的典型实验条件,1. Dimension (SCX) Column: SCX 5 um, 50 x 0.32mm Eluent A: 20 mM Citrat 25, % acetonitrile, pH 2.65 Eluent B: 500 mM NH4Cl in Eluent A, pH 2.65 Flow rate: 4 l/min Gradient: 0 bis 100 % B in 30 to 60 min2. Dimension (RPC) Trap-column: C18, 5 um, 5 x 0.3mm Column: C18, 3.5 um, 150 x 0.075mm Eluent A: 0.1% formic acid Eluent B: 0.1% formic acid in 84% acetonitrile Flow rate: 200 nl/min Gradient: 0.25 to 1% acetonitrile/min,LC的上样量,实验室规模 4.6 mm ID RPC column 0.2mg 2.1mm ID RPC column 0.250 g 0.075mm ID RPC column 0.05g 0.15mm ID RPC column 0.2 g Mono Q 10/10 50 mg 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!,Tandem MS,Data dependent MS/MS,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 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。,自动LC/MS/MS肽序列测定,+,多肽的裂解,碎片离子的命名,Residue name (A-Z) monoisotopic mass average massA 71.03711 71.0788B 114.53493 114.59625C 103.00919 103.1388D 115.02694 115.0886E 129.04259 129.1155F 147.06841 147.1766G 57.02146 57.0520H 137.05891 137.1412I 113.08406 113.1595J 0.0 0.0K 128.09496 128.1742L 113.08406 113.1595M 131.04049 131.1925N 114.04293 114.1039O 0.0 0.0P 97.05276 97.1167Q 128.05858 128.1308R 156.10111 156.1876S 87.03203 87.0782T 101.04768 101.1051U 150.95364 150.034V 99.06841 99.1326W 186.07931 186.2133X 111.0 111.0Y 163.06333 163.1760Z 128.55059 128.62315,氨基酸残基质量,CID条件下tryptic peptides裂解规律 mobile proton model,在低能CID条件下，tryptic peptides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，因此该类多肽的MS/MS图谱的base peak一般为母离子脱去修饰基团后产生的,MS/MS 图谱举例,Phosphorylated peptide MS/MS example,LPSIS*DLDS*IFGPVLSPK,Prominent loss of phosphoric acid from M+2H2+ ion,ESI-MS/MS vs MALDI-MS/MS why LTQ/LCQ type instrument so popular in proteomics?,数据库检索鉴定蛋白的过程,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SequestMascot,Finnigan Ion Trap Product Portfolio Technology you can trust,Built on innovations Proven by our customers,PERFORMANCE,Exceptional coverage,High sensitivity MSn,Finnigan LTQ FT,Ultimate Performance,Redefining Accurate Mass,Finnigan LTQ Orbitrap,Redefining Performance,Blackler, 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 positives,Finnigan LTQ FT Ultra High Performance,Ion Trap based Fourier Transform ICR Mass Spectrometer FTICR (FTMS) at LC timescale Accurate Mass High Sensitivity Ultra-high Resolution Above All - Simple to Operate,LTQ Orbitrap High Performance,Advanced LC-MS Proteomics Metabolomics Pharmaceutical Drug Discovery Small Molecule and Structure Elucidation Ultra-trace Level Analysis,Thermos New ETD System,Software for Proteomics,Biomarker Research,Robotics,Centrifuges,Concentrators,Liquid Chromatography/ Mass Spectrometry,Protein Identification,Protein Digest,Sample Handling,Data Analysis,Peptide Separation,Data Storage,Lab Information Management System,Microplate Readers,Protein Fractionation,Integrated Workflows,Freezers,Quantitative Proteomics and Biomarker Discovery and Validation,Company Confidential,A Word About Protein Quantification,Its a jungle out there,SILAC,AQUA,cICAT,iTRAQ,Isotope Labels,Isotope- Coded Tags,ICAT and iTRAQ are registered trademarks of Applera Corporation AQUA is a registered trademark of Harvard University,Thermo Supports ALL Quantification Methods,Isotope ICAT, cICAT, iTRAQ Tags Global, Complex, Expensive, Non-parallel workflowLimited practical success Isotope SILAC (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 interestBest sensitivity, accuracy and precisionMethod of Choice for Biomarker ValidationLabel- MS Peak Area Ratios Free Global, “Free”Method of Choice for Biomarker Discovery,A List of Putative Markers - What Next?,Validation Rather than continue to look for all proteins,develop a method to look only at a list of targeted proteins. Immunoassay? Multiplexed MS analysis of Labeled Peptides? Structural Characterization Exact protein isoform Top Down analysis,The “Peptide Hypothesis”,For 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, based 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 Aebersold,PROTEIN-AQUA in Practice,Identify Proteotypic Peptides for proteins of interest Order/synthesize isotopically enriched internal standards for these peptides Spike a known amount of the labeled peptide(s) into sample Digest sample Perform 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,Performance,Mass range m/z 30-3000 300 H-SRM transitions Multi-residue screening methods FAIMS enabled (food residues),Top Down Protein Analysis,Intact protein analysis determines exact protein isoform,Exactly What Protein Form is Present?,ETD: Electron Transfer Dissociation a New Fragmentation Mechanism for Proteomics,Organization of Eukaryotic DNA: Histones,Nuclear membrane,Chromatin fiber,Nuclear,pore,Nucleosomes,DNA,Chromatin fiber,(30 nm,dia,.),H1,Core,Histones,H3,H4,H2B,H2A,Nuclear membrane,Chromatin fiber,Nuclear,pore,Nucleosomes,DNA,Chromatin fiber,(30 nm,dia,.),H1,Core,Histones,H3,H4,H2B,H2A,adapted from Mathews et al., 3rd ed., 2000,Nuclear matrix,The N-terminal Tail of Histone H3,Methylation: mono-, di-, or trimethylation,Acetylation,Phosphorylation,M,P,Ac,H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50,M,P,M,P,P,P,P,Ac,Ac,Ac,LTQ/ETD allows alternating CID and ETD Scans,y,y,y,b,b,b,CID fragmentation,ETD fragmentation,z,z,z,c,c,c,ETD vs CID for MS/MS of Phosphopeptides,GRTGRRNsIHDIL 395 (M+4H)4+,CID MS/MS 395,ETD MS/MS 395,(M+3H - Phosphate)3+,蛋白质组学技术发展趋势,新型分离分析技术 新型生物质谱技术 定量蛋白组学技术 Top-down、middle-down、peptidome 膜蛋白 翻译后修饰 蛋白质组学技术在生命科学领域的应用 ,Thanks for your attention!,