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1、蛋白质组学的数据分析,邵晨,复习,蛋白质组的定义，蛋白质组学和基因组学的区别？ 由一个基因组，或一个细胞、组织表达的所有蛋白质。蛋白质组的概念与基因组的概念有许多差别，它随着组织、甚至环境状态的不同而改变。 在转录时，一个基因可以多种mRNA形式剪接，一个蛋白质组不是一个基因组的直接产物，蛋白质组中蛋白质的数目有时可以超过基因组的数目。,Key advantage of proteomics Researchers work on the level of gene products and deal with genes that are really expressed to give a
2、 detectable PRODUCT and are not just “expressed“ which only says they produce a detectable mRNA but it is not clear whether there is a gene product or not. Key limitation of proteomics Usually, only a fraction of the proteins synthesized can be detected in a proteomics experiment, whereas the expres
3、sion of ALL genes can be monitored in a whole-genome array experiment. Key prerequisite of proteomics A genome sequence for the investigated organism or at least a collection of many cDNA sequences is required.,From Yogita Mantri & Arvind Gopus presentation in 2003,蛋白质组学研究的目标,蛋白质鉴定 蛋白质特性-如翻译后修饰 蛋白质定
4、量-相对定量、绝对定量 样品间比较 定性-不同样品间含有的蛋白类型的差异 定量-不同样品间含有的蛋白浓度/含量的差异 翻译后修饰-不同样品间是否存在不同的翻译后修饰形式 蛋白质功能,把单个蛋白/多肽从复杂样品中分离出来非常困难，在“组学”实验中一般达不到这个效果,Ionization methods,Electrospray mass spectrometry (ESI-MS) Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse a
5、nalyte. Charge imparted from rapidly evaporating liquid. Matrix-assisted laser desorption ionization (MALDI) Analyte (protein) is mixed with large excess of matrix (small organic molecule) Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.,MALDI
6、m/z spectrum of a peptide mixture,The Quadrupole,The quadrupole consists of four parallel metal rods. Ions travel down the quadropole in between the rods. Only ions of a certain m/q will reach the detector for a given ratio of voltages: other ions have unstable trajectories and will collide with the
7、 rods. This allows selection of a particular ion, or scanning by varying the voltages.,source,detector,Voltage,Filters out all m/z values except the ones it is set to pass Obtains a mass spectrum by sweeping across the entire mass range,Collects and store ions in order to perform MS-MS analyses on t
8、hem.,Ion Trap Mass Analyzer,The trap consists of a top and a bottom electrode and a ring electrode around the middle.Ions are ejected on the basis of their m/z values.To monitor the ions coming from the source, the trap continuoulsy repeats a cylcle of filling the trap with ions and scanning the ion
9、s according to their m/z values.,Separates the mass analysis and ion isolation events in time (using a single mass analyzer),Ionization,ion transfer/trapping,parent ion isolation/ fragmentation,daughter ion detection,14,A mass analyzer for determining the mass-to-charge ratio (m/z) of ions based on
10、the cyclotron frequency of the ions in a fixed magnetic field.,All ions are detected simultaneously over some given period of time,Ions are injected into a magnetic field , that causes them to travel in circular paths. Excitation with oscillating electrical field increases the radius and enables a f
11、requency measurement,Fourier Transform MS,Fourier transform ion cyclotron resonance mass spectrometry, FTICMS,ICR can be used with different ionization methods, ESI, MALDI,A short sweep of frequencies is used to excite all ions. The complex spectrum of intensity/time is analyzed with Fourier Transfo
12、rm to extract the m/z componets,High resolution High accuracy Very sensitive (the minimal quantity for detection is in order of several hundered ions Non destructive the ions dont hit the detection plate so they can be selected for further fragmentation,Orbitrap 静电轨道阱质谱傅里叶变换原理,Mass Spectrometry Revi
13、ews，Volume 27,Issue 6,蛋白质组学研究的目标,蛋白质鉴定 Top-down策略（质量纹方法，MS谱图） Bottom-up策略（de novo测序和数据库检索，MS/MS谱图） 蛋白质修饰 蛋白质定量-相对定量、绝对定量 样品间比较 蛋白质功能,Top-down proteomics,一级质谱图,指纹数据库,http:/ Mass Fingerprinting，PMF）是从一级质谱（MS）中鉴定多肽的主要方法。多肽质量纹一般都用于分析2DE-MS的结果，不适宜分析多个蛋白质的混合物。,多肽质量纹鉴定,蛋白质经过酶解后，送入质谱仪，得到一级质谱，即多肽离子的m/z。 从一级质
14、谱鉴定蛋白质的算法主要用在MALDI-TOF产生的质谱图上。 目前来说，由MALDI-TOF质谱仪产生的质谱图精度较高。 另一个问题是，ESI产生的质谱图中的离子通常带有很多电荷，而MALDI质谱图中的离子一般只带一个电荷，比较容易计算。,蛋白序列数据库,质量纹算法的核心是将实验获得的蛋白指纹与数据库中的蛋白指纹进行匹配，为此，必须首先找到一个合适的蛋白质序列数据库在网上可以查询到最新的蛋白序列数据库，如NCBI，UniProt, SwissProt等等 下载FASTA格式,Protein sequence database,Uniprot（包含Swissprot和Tremble）,Integ
15、r8,FASTA格式的数据库,FASTA格式包含蛋白的名称和氨基酸序列。,虚拟酶解,有了蛋白序列的信息，我们就可以进行鉴定。 对应于送进质谱仪的样品，首先找到数据库里的序列的酶切位点。,质量排列,这样可以产生一系列的多肽，我们可以计算每个多肽的分子量。最后一个R的质量多加了18，这是因为我们写在下面的是残基的分子量。,肽和肽键,质量排列,把所有多肽的分子量排序。,质量纹,如此，质谱图上的质量就可以与多肽上的质量相匹配。,http:/ m/z: 1019.08,解决方案,第一个解决的办法是限制用来搜索的数据库。比如，你如果做的试验用的是小鼠的组织，那么你可以只在小鼠的数据库中搜索，这样就可以减低出现这种情况的可能性。第二个解决的办法是要求必须有多个多肽和数据库相匹配，才做出最后的蛋白质鉴定。,多匹配,DFPIANGER 1019.09,EPISVSSQQMLK 1347.56,VLDALDSIK 974.13,Carbonic anhydrase II,
