2018/6/11 蛋白質體學 Proteomics 2016 Mass Spectrometry 陳威戎 2016. 11. 28.

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1 2018/6/11 蛋白質體學 Proteomics 2016 Mass Spectrometry 陳威戎

2 Why mass spectrometry? Fast High throughput Accuracy Many Applications

3 Mass Spectrometry 1. Mass spectrometer compositions
2018/6/11 Mass Spectrometry 1. Mass spectrometer compositions 2. Mass spectrum at a glance 3. Difficulty of sample ionization 4. Matrix Assisted Laser Desorption/Ionization (MALDI) 5. Electrospray Ionization (ESI) 6. Mass analyzer types- TOF, Q, Iontrap 7. Type of mass spectrometry 8. MALDI-TOF MS vs. LC-ESI-Q-TOF MS/MS

4 Mass spectrometer composition

5 Mass spectrum at a glance
A. The Y axis is relative intensity B. The X axis is m/z C. The tallest peak in the spectrum: "base peak“ D. The counts associated with the tallest peak in the spectrum E. Other peak(s)

6 Difficulty of sample ionization
Solid phase  Gas phase polar, non-volatile molecules : kDa gas phase “Soft” Methods: Matrix Assisted Laser Desorption Ionization (MALDI) Electrospray Ionization (ESI) Ionisation methods include the following: Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Electrospray Ionisation (ESI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Matrix Assisted Laser Desorption Ionisation (MALDI) Thermospray Ionisation (TSP) The ionisation methods used for the majority of biochemical analyses are Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI), and these are described in more detail in Sections 6 and 7 respectively. With most ionisation methods there is the possibility of creating both positively and negatively charged sample ions, depending on the proton affinity of the sample. Before embarking on an analysis the user must decide whether to detect the positively or negatively charged ions.

7 Matrix Assisted Laser Desorption Ionization (MALDI)
Animation

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12 Matrix Selection

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16 Electrospray ionization (ESI)
Generates ions directly from acidic solution The production of ions by evaporation of charged droplets obtained through spraying or bubbling, has been known about for centuries, but it was only fairly recently discovered that these ions may hold more than one charge4. A model for ion formation in ESI, containing the commonly accepted themes, is described below5: Large charged droplets are produced by 'pneumatic nebulization'; i.e. the forcing of the analyte solution through a needle (see figure), at the end of which is applied a potential - the potential used is sufficiently high to disperse the emerging solution into a very fine spray of charged droplets all at the same polarity. The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface. Eventually, at the Rayleigh limit, Coulombic repulsion overcomes the droplet's surface tension and the droplet explodes. This 'Coulombic explosion' forms a series of smaller, lower charged droplets. The process of shrinking followed by explosion is repeated until individually charged 'naked' analyte ions are formed. The charges are statistically distributed amongst the analyte's available charge sites, leading to the possible formation of multiply charged ions under the correct conditions. Increasing the rate of solvent evaporation, by introducing a drying gas flow counter current to the sprayed ions (see figure), increases the extent of multiple-charging. Decreasing the capillary diameter and lowering the analyte solution flow rate i.e. in nanospray ionization, will create ions with higher m/z ratios (i.e. it is a softer ionization technique) than those produced by 'conventional' ESI and are of much more use in the field of bioanalysis.

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20 Electrospray ion generation
Cys+Ser+Arg

21 Nanospary ionization Flow Rates: Reduce the sample amount
ESI: 100 ml/min Nanospary: nl/min Reduce the sample amount Increase the analysis time

22 Mass analyzer types Time of flight (TOF) Quadrupole Ion-Trap
Consideration: Accuracy Mass range

23 Time-of-Flight (TOF) Smaller ions faster
Development Time-of-flight mass spectrometry (TOF-MS) was developed around fifty years ago and the first commercial instrument was marketed by the Bendix Corporation in 1955 based on the Wiley and McLaren design1. TOF-MS has only recently begun to fulfil its true potential with the development of higher resolution instruments. The inherent characteristics of the TOF mass analyser, lead to spectra of virtually unlimited mass range being obtained in a few microseconds with relative ease. Recently, along with the introduction of matrix-assisted laser desorption/ionization in 1988 there has been a large increase in interest in TOF-MS, especially in the fields of biological and polymer chemistry. Basic Theory of TOF-MS The TOF mass spectrometer (see figure) is the simplest type of common mass analyser and has a very high sensitivity at a virtually unlimited mass range. The sample ions are generated in a source zone, s, of the instrument, by whatever ionization method is being employed. A potential, (Vs - the source extraction) is applied across the source to extract and accelerate the ions from the source into the field-free 'drift' zone of the instrument, d. In the ideal case, all ions produced will leave the source at the same time with the same kinetic energy, due to their having been accelerated through the same potential difference. In this case the time-of-flight of the ions produced will only be dependent on the mass and the charge of the produced ion. Neglecting the extraction time from the source, the basic formula for TOF mass analysis is given by the equation:   Where: mi = mass of analyte ion zi = charge on analyte ion E = extraction field ti = time-of-flight of ion ls = length of the source ld = length of the field-free drift region e = electronic charge (1.6022x10-19 C) For a reliable mass spectrum to be obtained, the time of ion extraction must be known to a high degree of accuracy. This problem is usually addressed by using a pulsed ionization technique like laser desorption or MALDI. There are a number of problems with the technique which cause a time-of-flight distribution at each mass, thus lowering the resolution2. These factors must be corrected or allowed for if a high-resolution spectrum is required. Achieving high resolution normally involves using the more complex reflectron instruments3, long flight tubes and/or delayed ion extraction. Smaller ions faster high sensitivity, unlimited mass range Accuracy: MALDI Resolution: reflection instruments Animation

24 Reflection TOF Animation

25 Quadrupole mass analyzer
+(U+Vcos(wt)), -(U+Vcos(wt)) U: fixed potential Vcos(wt): radio frequency (RF) field of amplitude V and frequency w The four rods are shown as being circular in the diagram but in practice they have a hyperbolic cross-section. Two opposite rods will have a potential of +(U+Vcos(wt)) and the other two -(U+Vcos(wt)) where U is a fixed potential and Vcos(wt) represents a radio frequency (RF) field of amplitude V and frequency w. When cos(wt) cycles with time, t, the applied voltages on opposed pairs of rods will vary in a sinusoidal manner but in opposite polarity (due to them being offset). Along the central axis of the quadrupole assembly and also the axis between each adjoining rod the resultant electric field is zero. In the transverse direction of the quadrupoles, an ion will oscillate amongst the poles in a complex fashion, depending on its m/z, the voltages U and V and the frequency, w, of the alternating RF potential. By suitable choices of U, V and w, only ions of one m/z will oscillate stably through the quadrupole mass analyser to the detector. All other ions will have greater amplitude of oscillation causing them to strike one of the rods. In practice, the frequency w is fixed with typical values being 1-2 MHz. The length and diameter of the rods will determine the mass range and ultimate resolution that can be achieved by the quadrupole assembly. However, the maximum mass range that is normally achieved is around 4000Da with a resolution of around 2000.

26 Ion trap analyzer Components: 3D quadrupolar potential field
ring electrode entrance endcap electrode exit endcap electrode 3D quadrupolar potential field Trajectory: trapping potential and the m/z of the ions advantages: multiple CID without multiple analyzers compact size trap and accumulate ions to increase the signal-to-noise ratio

27 Nobel Prize Winners related to MS

28 Type of Mass Spectrometry
MALDI-TOF SELDI-TOF MALDI-Quardrupole-TOF ESI-Quardrupole-TOF ESI-Triple Quardrupole ESI-Iontrap

29 MALDI-TOF Mass Spectrometry
Reflectron Detector Sample plate

30 Quardrupole-TOF Mass Spectrometry

31 Nanospray ESI-Q-Iontrap Mass Spectrometry

32 Thermo Finnigan LCQ-Deca
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33 Interpreting Electrospray Mass spectra - Calculating mass
Charge State Calculation Unprotonated Mass +1 379.2 x 1 - 1 378.2 +2 190.1 x 2 - 2 33

34 Isotopic Effect

35 Multiple charged state
Lys and Arg (M+nH)n+ in positive ionisation mode The m/z values can be expressed as follows:

36 Theoretical Mw of hen egg lysozyme (based on average atomic masses): 14305.1438 Da.

37 Mass spectra comparison between MALDI-QTOF and ESI-QTOF

38 Protein identification

39 蛋白質體學研究工作站 MALDI-TOF MS Picker & Digestion robotics LC-Triple Quad MS
Database Search Workstation

40 質譜儀 (MS) MALDI： Matrix-assisted laser desorption/ionization
基質輔助雷射脫附/離子化 固態樣品 優點: 對鹽類耐受度高 缺點: 僅生成單電荷離子, 不易對大分子進行分析 ESI： Electrospray ionization 電噴灑法離子化 液態樣品 優點: 可產生穩定的複數帶電離子, 降低質荷比(m/z), 有利於巨大分子之分析 缺點: 對鹽類耐受度低

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43 基質輔助雷射脫附離子化質譜儀 (MALDI-TOF MS)
純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

44 液相層析電灑法離子化串聯式質譜儀 (LC-ESI MS/MS)
純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

45 蛋白質身份鑑定- 胜肽質量指紋 (PMF) 純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

46 Protein identification by peptide mass fingerprinting (PMF)

47 蛋白質身份鑑定- 胜肽質量指紋 (PMF) Gel Database Protein ? 1 2 3 tryptic
digestion Gel Database ? 1 2 3 MS Analysis stored data or theoretical peptides “ 純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

48 蛋白質身份鑑定- 串聯式質譜定序法 (MS/MS)
純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

49 Protein identification by MS/MS

50 4700 Proteomics Analyzer, Applied Biosystems
Mass analyzer Tandem MS Ion source Detector Basic components of any MS 4700 Proteomics Analyzer, Applied Biosystems 50

51 MS, followed by precursor ion selection
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52 Fragment ion spectrum Tandem MS 52

53 Tandem mass spectrum 53

54 Tandem mass spectra (MS/MS) can be used for peptide sequencing
Database Searching Peptide Mass Fingerprinting Sequence tag approach De novo sequencing inspect raw data 54

55 蛋白質身份鑑定- 串聯式質譜定序法 (MS/MS)
純化酵素是一件非常基本的工作，很多重要的研究，都脫不開酵素的純化工作。而大多數酵素的純化，基本上也脫不開一些最基本的原則。 首先，建立一個完善的酵素實驗室是很必要的；我們把許多實驗室內的運作細節一一交代，期望同學能認知這些經驗，確實接收並養成習慣，且期望應用到將來每個人的研究工作上。 最先遇到，但是最容易被忽視的步驟，就是材料處理及總蛋白質的抽取。將提醒你小心選擇採料的種類、時期、部位等，並選擇一個良好的粗抽取方式，以便有一個良好的開始。

56 Peptide Sequence from CID: b, y series