蛋白质组学的数据分析
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。
MALDI m/z spectrum of a peptide mixture
The Quadrupole
source
detector
The quadrupole consists of four parallel metal rods. Ions travel down the quadropole in between the rods.
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
To monitor the ions coming from the source, the trap continuoulsy repeats a cylcle of filling the trap with ions and scanning the ions according to their m/z values.
► 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 Gopu’s presentation in 2003
► Key advantage of proteomics
▪ Researchers work on the level of gene products and deal with genes that are really expressed to give a 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.
► 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.
This allows selection of a particular ion, or scanning by varying the voltages.
Voltage
Filters out all m/z values except the ones it is set to pass
ObtБайду номын сангаасins a mass spectrum by sweeping across the entire mass range
► Key limitation of proteomics
▪ Usually, only a fraction of the proteins synthesized can be detected in a proteomics experiment, whereas the expression of ALL genes can be monitored in a wholegenome array experiment.
Ion Trap Mass Analyzer
Ions in
Trapped ions
Ions out
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.
Ionization methods
► Electrospray mass spectrometry (ESI-MS) ▪ Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid.
Collects and store ions in order to perform MS-MS analyses on them.
Separates the mass analysis and ion isolation events in time (using a single mass analyzer)
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 rods.