质谱数据分析2014
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► 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.
Collects and store ions in order to perfor百度文库 MS-MS analyses on them.
Separates the mass analysis and ion isolation events in time (using a single mass analyzer)
► 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.
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.
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
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。
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.
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.
MALDI m/z spectrum of a peptide mixture
The Quadrupole
source
The quadrupole consists of four parallel metal rods. Ions travel down the quadropole in between the rods.
► 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.
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.
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
Obtains a mass spectrum by sweeping across the entire mass range
► 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
▪ 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.
Collects and store ions in order to perfor百度文库 MS-MS analyses on them.
Separates the mass analysis and ion isolation events in time (using a single mass analyzer)
► 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.
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.
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
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。
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.
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.
MALDI m/z spectrum of a peptide mixture
The Quadrupole
source
The quadrupole consists of four parallel metal rods. Ions travel down the quadropole in between the rods.
► 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.
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.
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
Obtains a mass spectrum by sweeping across the entire mass range
► 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