Mass Spectrometer (MS) For proteomic research

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Mass Spectrometer (MS) For proteomic research

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What does a mass spectrometer do?

1. It measures mass better than any other technique.

2. It can give information about molecular weight and chemical structures.

What are mass measurements good for?

To identify (鑑定), verify (證明), and quantitate (定量):

metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

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Important performance factors for MS analysis

Mass accuracy: How accurate is the mass measurement?

Resolution: How well separated are the peaks from each other?

Sensitivity: How small an amount can be analyzed?

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Pharmaceutical analysis Bioavailability studies

Drug metabolism studies, pharmacokinetics Characterization of potential drugs

Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization

Proteins and peptides Oligonucleotides Environmental analysis

Pesticides (殺蟲劑) on foods Soil and groundwater contamination Forensic (法庭) analysis/clinical

Applications of Mass Spectrometry (MS)

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The advantage of MS vs. Protein sequence 1. High sensitivity

2. Fast analysis 3. More information

4. Post-translational modification (PTM)

The application of MS in proteomic 1. Protein ID

2. Protein PTM 3. Protein quantity

4. Protein-protein interaction

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Mass spectrometer vs. proteomic

MALDI(Matrix-assisted Laser Desorption lonization): Tanaka 1987 ESI(Electrospray ionization): Dr. John B. Fenn 1988

Nobel Prize in Chemistry 2002

"for the development of methods for identification and structure analyses

of biological macromolecules"

"for their development of soft desorption ionisation methods for mass spectrometric

analyses of biological macromolecules"

John B. Fenn Koichi Tanaka

ESI MALDI

b. 1959 b. 1917

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Overview of Mass Spectrometry

Mass Spectrum

Mass Analyzer Ionization

M+/Fragmentation Sample

Molecule (M)

Valuable information by MS:

1. Element analysis 2. Molecular weight 3. structure 4. Mixture compound

質譜元件:

進樣系統 (分離) → 離子源 → 質量分析器 → 偵測器 → 電腦分析 m/z mass-to-charge ratio 質荷比

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The basic structure of MS (for proteomic)

Sample enter system

Ionization

analyzer

Detector

Data analysis

High performance liquid chromatography; HPLC Capillary electrophoresis; CE

MALDI ESI

TOF (Time of flight) 飛行時間 Quadrupole 四極棒式 Ion-trap 離子阱

離子 → 電極板 →電子→ 訊號強度… Vacuum

9 Ion source Mass analyzer Detector

Ion generation Ion separation Ion detection

F. Lottspeich and H. Zorbas, Bioanalytik 1998, Spektrum Akad. Verlag

Mass spectrometry determines the molecular weightof chemical compounds by separating molecular ions in a vacuum according to their mass-to-charge ratio (m/z)

Ions are generated by induction of either the loss or the gain of a charge (protonation, deprotonation or electron injection)

Generated ions can be fragmented in the vacuum, and the resulting sub- fragments can provide information about the structure of a compound

Basics of Protein Mass Spectrometry

Inlet Ion source

Mass

Analyzer Detector Data System High Vacuum System

Mass Spectrometer Block Diagram

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Mechanisms of Sample Ionization

• Protonation: M + H

MH

• Cationization: M + Cat

MCat

• Deprotonation: MH M

+ H

• Electron Ejection: M M

^. + e-

• Electron Capture: M + e- M

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電子轟擊電離 Electron Impact Ionization, EI 化學離子化 Chemical Ionization, CI

場電離，場解吸 Field Ionization FD, Field Desorption FD 快原子轟擊 Fast Atom Bombardment, FAB

基質輔助雷射解析電離 Matrix-Assisted Laser Desorption Ionization, MALDI 電噴霧電離 Electrospray Ionization, ESI

大氣壓化學電離 Atmospheric Pressure Chemical Ionization, APCI

質譜分析第一步:樣品必需離子化 (ionization)

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Ionization (ion source)

MS analysis must form the ionization of sample to gas phase

<1980, heat → sample →aeration(氣化) →ionization

1980, FAB; fast-atom bombardment, < 3000 Da(protein analysis possible)

1985, plasma desorption, <45000 Da; but long time need 1987, 1988 MALDI and ESI (soft ionization, protein contact)

1987 Koichi Tanaka MALDI 1988 John B. Fenn ESI

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電子轟擊電離又稱為電子轟擊，或電子電離。是應用最 普遍，發展最成熟的電離方法。

轟擊電壓 50-70eV, 有機分子的電離電位一般為7-15eV。

可提供豐富的架構訊息。

有些化合物的分子離子不出現或很弱

電子轟擊電離 EI

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+

+ +

+

: R2 : R3 : R4 : e

+

M⁺ (M-R₂)⁺ (M-R₃)⁺

Mass Spectrometer (M-R₁)⁺

電子轟擊電離 Electron Impact（EI）

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離子室內的回應氣（甲烷等；10~100Pa，樣品 的103~105倍），電子（100~240eV）轟擊，產生離 子，再與試樣分離碰撞，產生準分子離子。

最強峰為準分子離子；

譜圖簡單；

不適用難揮發試樣；

+

+

氣体分子 (甲烷)

樣品分子

+ ^{準分子離子 電子}

(M+1)⁺;(M+17) ⁺;(M+29) ⁺;

化學電離

（Chemical Ionization，CI）

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Ionisation Methods: Chemical Ionisation (CI)

»0.5 mmHg (low pressure, but high volume) Bimolecular collisions (碰撞)

CH5⁺behaves as “super acid＂

CH

^.

CH

CH

+ CH

^.

CH₅⁺protonates sample: e.g.

R₃C-OH +CH₅⁺ R₃C-⁺OH₂ +CH₄ Detected

Mass detected is [M+H]⁺

‧Not a radical cation: c.f. EI EI & CI: applicable to low MW analytes i.e less than approximately 800 Daltons

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場致電離 Field Ionization, Field Desorption（FI，FD）

電壓︰7-10 kV；d<1 mm；

強電場將分子中拉出一個電子；

分子離子峰強；

碎片離子峰少；

不適合化合物架構監定；

鉑板 +

+ + +

+ +

+ +

+ +

+ +

+

d<1mm 鉑板

當樣品蒸汽接近或接觸帶高正電位的金屬針時，

在強電場的作用下發生電離。要求樣品分子處于氣 態，靈敏度不高，應用逐漸減少。

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樣品不需汽化, 預先附在處理好的場離子發射 體上, 送入離子源, 然後通以微弱電流, 使樣品分子 從發射體上解吸下來, 並擴散至高場強的場發射區, 進行離子化。

適用于難汽化, 熱不穩定的樣品. 如: 糖類，

類，金屬有機化合物，聚合物等。

場解吸 FD

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惰性氣體Ar或Xe的原子首先被電離並被加速， 使之具 有高的動能，在原子槍(atom gun)內進行電荷交換回應︰

Ar⁺（高動能的）+ Ar（熱運動的）→Ar（高動能的）+ Ar⁺（熱運動的）

高動能的Ar或Xe原子束再轟擊樣品分子使其離子化

快原子轟擊

Fast Atom Bombardment（FAB）

FAB是目前廣泛使用的軟電離技術，適用于難汽化,極性強的大分 子。樣品用基質調節后黏附在靶物上。

常用的基質有甘油，硫代甘油，3-硝基 醇，三乙醇胺等。

注意︰FAB質譜圖中會出現基質分子產生的相應的峰及基質分 子與樣品分子的結合峰。

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不可揮發

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MALDI可使熱敏感或不揮發的化合物由固相直接得到 離子。

待測物質的溶液與基質的溶液混合後蒸發，使分析物 與基質成為晶體或半晶體，用一定波長的脈波式雷射進行 照射時，基質分子能有效的吸收雷射的能量，使基質分子 和樣品分子進入氣相並得到電離。

MALDI適用於生物大分

子，如蛋白質類，核酸類

化合物。可得到分子離子 峰，無明顯碎片峰。此電離 模式特別適合于飛行時間質 譜計。

基質輔助雷射解析電離

Matrix-Assisted Laser Desorption Ionization (MALDI)

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Ionisation Methods:

MALDI

‧Matrix Assisted Laser Desorption Ionisation

‧Solid matrix: e.g. dihydroxybenzoic acid

‧Matrix absorbs energy at l of laser used

‧Causes vapourisation (汽化) of matrix and analyte

‧Proton transfer to analyte (or Li, Na, K)

‧Detection by mass spectrometer

‧Applicable to MW up to 12,000D and

‧Thermally labile (不穩定) analytes

‧Polymers and biomolecules

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Pulsed laser beam

Sample ions

Ion analyser

Sample plate

Extraction grid Sample, matrix and cations Pulsed laser

beam

Sample plate

Ionisation Methods:

Schematic of A MALDI Chamber

‧Solid solution

‧Matrix/analyte/cations

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介質輔助雷射脫附離子化(matrix-assisted laser desorption ionization; MALDI)

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Laser

Laser 如何藉由如何藉由MATRIX 使MATRIX 使Protein Ionization？Protein Ionization？

Matrix 的特性 ：

可吸收 UV wavelength 的 Laser 光源，並增加能量 除釋放熱能外，並可游離 H⁺，使得 Protein 接受，並帶有

電荷 （M+H⁺）

Protein +H⁺ 在真空狀態下，霧化形成氣態單一分子

（Plume Gas）

在高壓電場的推動下 Protein 開始朝負極飛離

• L

a s e r

Matrix Protein/Peptide

Time of Time of Flight Flight Mass Mass Spect Spect

Matrix - CHCA

Bruker Daltonics, Taiwan Office

27 Laser → energy → matrix → photochemical reaction (UV) →

protonated matrix → interaction with peptide → proton transfer → gas phase protonated peptide → m/z

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蛋白質用介質輔助雷射脫附離子化質譜儀分析所能得到的數據 蛋白質樣本經介質輔助雷射脫附離子化過程會形成帶有正電荷的氣態蛋 白質離子，正電荷也是以質子化（protonation）的方式形成於蛋白質的 鹼性官能基（basic functional group）上。不過，經介質輔助雷射脫附 離子化過程形成氣態蛋白質離子，蛋白質樣本經電噴灑離子化過程一般 情形下形成帶有多個正電荷的氣態離子，鹼性氨基酸數目越多則氣態蛋 白質離子電荷數目越大；相對的，不論鹼性氨基酸數目多寡，蛋白質樣 本經介質輔助雷射脫附離子化過程一般情形下形成只帶有一個正電荷的 氣態離子為主，有時也會觀察到帶有兩個、甚至三個的正電荷的氣態離 子，但是它們的訊號強度多半較只帶有一個正電荷的氣態離子低。

牛血清白蛋白（BSA）樣本 經介質輔助雷射脫附離子化 所產生的質譜圖譜，其中標 示的訊號代表帶一個與兩個 正電荷數目的蛋白質離子。

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1. Isolation of the biomolecules from each other

• The biomolecules are incorporated (結合) in a large excess of matrix molecules, strong intermolecular forces are thereby reduced (matrix isolation). protein經 由跟matrix結合,使protein 間作用降低

• One of the most important conditions for matrix to work well is an intimate (最理想) mixing of the analyte and the matrix.

• optimal molar ratios are in the 1:1000 to 1:10000 (analyte to matrix) range. This means that analyte molecules are singularly (非常地) and fully surrounded (solvated) by matrix molecules.

The matrix serves three major functions

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2. Absorption of energy from the laser light

• The matrix molecules absorb the energy from the laser light and transfer it into excitation energy of the solid system.

– Thereby inducing an instantaneous (即時) phase transition of a small molecular layers of the sample into a gaseous species.

3. Ionization of the biomolecules

• An active role of the matrix in the ionization of the analyte molecules by photoexcitation or

photoionization of matrix molecules, followed by proton transfer to the analyte molecules is likely, though not proven unequivocally (明確地) to date.

• In practice, success or failure of a matrix depends on properties other than acting as an energy transfer mediator

The matrix serves three major functions

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The most commonly used Matrix for MALDI

Matrix Wavelength Comments

2,5-Dihydroxy benzoic acid (DHB) 337

DHB + 10% 5-methoxy salicylic acid 337 used for masses >20 kDa Nicotinic acid 266 nm

4-Hydroxy picolinic acid 337 used for oligonucleotides

Glycerol 2.94μm liquid matrix

U. Bahr, M. Karas, F. Hillenkamp, Fresenius J. Anal. Chem. 348, 784 (1994).

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Select matrix

Prepare matrix

Prepare sample

Mix sample and matrix

Load sample on clean sample plate

Dry

Sample Preparation for MALDI

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Selection of Matrix

Sinapinic Acid Proteins >10kDa a-Cyano-4-Hydroxy-

cinnamic acid

Peptides<10kDa 2,5 Dihydroxybenzoic

acid

Neutral Carbohydrates, Synthetic Polymers

“Super DHB” Proteins,

Glycosylated proteins acid

Oligonucleotides HABA Proteins,

Oligosaccharides 3-Hydroxypicolinic

35 Compatible 可相共處 36

37 38

流出液在高電場下形成帶電噴霧，在電場力作用下穿過氣 帘；

氣帘的作用︰霧化；蒸發溶劑；阻止中性溶劑分子

電噴霧電離（ESI）

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Electrospray: Generation of aerosols and droplets 浮質

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電噴灑離子化(Electrospray Ionization; ESI)

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電噴灑離子化(Electrospray Ionization; ESI)

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+

+ + +

+

+ + +

+

-

-

- -

- - -

- -

+

+ + + + + ++ +

+ + + + + +

+ + + + + + + + + + + + + + + + ++ +

+ +

+ +

+ +

+ + ++

+ + + ++

+ + + + +

+ + +

+ + + + + +

+ + + + + + + +

+ + + + + +

+ +

+ +

+ +

+ +

+ + +

+ +

+ +

+

+ +

+ +

+ + +

+ +

+ + +

+ +

+ + +

+ +

+

Cathode

_ + Power Supply

+ +

+ +

+ + +

+ +

+

+ +

+ + + +

+

+ +

Electrospray process

• Analyte dissolved in a suitable solvent flows through a small diameter capillary tube

• Liquid in the presence of a high electric field generates a fine “mist” or aerosol spray of highly charged droplets

10⁶charges for 30 micron droplet

+ -

44 High voltage applied

to metal sheath (~4 kV)

Sample Inlet Nozzle (Lower Voltage)

Charged droplets

++

+++ + ++

+ +++ ++ + +++ +++

+++ +++

+ + + +

+ + +

+ + + +++

+++ ++ +

MH⁺

MH₃⁺ MH₂⁺ Pressure = 1 atm

Inner tube diam. = 100 um

Sample in solution N₂

N2 gas

Partial vacuum

Electrospray ionization:

Ion Sources make ions from sample molecules (Ions are easier to detect than neutral molecules.)

45 Rayleigh

Limit Reached

+ + +

+ + +

+ + +

- +

--- -

+ ++ + +

+ + + +

-+ ----

+ +

+ ++

+--

+ + + --+ ++

Evaporation

+

+

Charged Droplets

樣品離子 准分子离子

Analyte Ions

Solvent Ion Clusters Salts/Ion pairs

Neutrals

++

+ +

+ --+ ++ 其他離子

電噴霧電離（ESI）

-

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ESI vs. m/z

One peptide

↓ ESI

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利用電噴灑離子化質譜儀分析蛋白質時，常在蛋白質樣本溶液中加入甲酸

（formic acid）或乙酸（acetic acid）等易揮發的酸，來幫助氣態蛋白質正 離子的產生。利用甲酸製備酸化的蛋白質樣本溶液，然後經電噴灑形成帶 有多個正電荷的氣態蛋白質離子過程，可以利用下列化學式來說明。

solvation electrospray

M _(solid) + H₂O+HCOOH [M+mH]ⁿ⁺_(liquid) [M+nH]ⁿ⁺_{( gas )}（式一）

其中，M代表蛋白質分子，m是溶液中蛋白質帶正電荷數目，n是氣態蛋白質離 子的電荷數目，電荷數目一定是整數；m與n的大小不一定相同，但與蛋白質鹼 性氨基酸數目及酸的強度（受酸的種類、濃度、溶液組成等因素影響）

質譜儀分析是一種氣態離子的質荷比（mass to charge ratio, m/z）量測，對 上述以質子化方式形成的氣態蛋白質離子而言，質量是蛋白質分子量加上n 個質子酸，質子酸（H+）的質量是1.0007（約為1），電荷數目是n（整 數）。

質荷比（m/z） = (M+n)/n （式二）

其中，M在此代表蛋白質分子量；n是電荷數目（整數）；m/z是質荷比。

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電噴灑時因產生的眾多氣態蛋白質離子所帶的正電荷數目不同會呈現一個分 佈。換句話說，同時電噴灑所產生的眾多氣態蛋白質離子，有些帶的電荷數目 多一點，有些帶的電荷數目少一點，呈現一個分佈。圖是溶菌酶蛋白

（lysozyme）經電噴灑產生的質譜圖譜，其中的訊號代表帶不同正電荷數目 的蛋白質離子。電噴灑離子化質譜儀能精確量測蛋白質分子質量，那麼，由圖 六的質譜圖譜如何計算蛋白質分子量呢？圖六中的訊號都已經標示了蛋白質離 子帶正電荷的數目，利用（式二）即可計算出蛋白質的精確質量。以訊號強度 最強的m/z 2044.7訊號峰為例：

質荷比（m/z） = (M+n)/n

將圖六的數據帶入上式，得2044.7 = (M + 7)/7即可計算出蛋白 質的精確質量為 14306 Da

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每一個訊號峰都可以據以計算出蛋白質的精確質量，當然，其先決條件是 必須知道產生該訊號峰的蛋白質離子之帶正電荷的數目。然而質譜圖數據只 能提供質荷比，那要如何知道每個訊號峰所對應的電荷數目呢？常用的方式 有兩種。首先想像圖六中的訊號峰都未標示電荷數目，第一種方式假設圖六 中相鄰的訊號峰所對應的電荷數目相差1；我們知道，電荷數目都是整數，同 時對某一個蛋白質離子而言，帶正電荷的數目越大，所產生的訊號峰質荷比 越小，所以利用（式二）與圖六中的兩個相鄰的訊號峰 m/z 2044.7及m/z 1789.0，可以得到下列聯立方程式：

(1) 2044.7 = (M + n)/n (2) 1789.0 = (M + n+1)/(n+1)

解開上述聯立方程式中的兩個未知數M與n，同時考慮n必須是整數的限 制，即可計算出電荷數目n值與蛋白質的精確質量M值。圖六中的每一個訊號 峰都可以利用相似的過程計算出一個蛋白質的質量，不同訊號峰計算出的值 可能因訊號峰m/z值的量測不確定度而稍有差異，可以利用求平均值的方式 減小量測不確定度。

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第二種知道訊號峰所對應之帶正電荷數目的方式是利用同位素訊號峰，此種 方式一般應用於分子量較小的蛋白質，以下利用圖來說明。

圖是一個1178 Da的胜肽（peptide）所產生的電噴灑質譜圖譜，可以看到有兩群訊號峰；右 邊的訊號峰群是由帶1個正電荷的胜肽所產生，訊號峰群中有m/z 1178.7、1179.7、

1180.7、1181.7等數值連續相差1.0的四個訊號峰，這些訊號峰是由自然界中的同位素分布 所造成的。左邊的訊號峰群是由帶2個正電荷的胜肽所產生，m/z值應為(M+2)/2，所以可以 看到m/z 589.9、590.4、590.9、591.4等數值連續相差0.5的四個訊號峰。反過來說，如果 訊號峰所對應的電荷數目未知，查看同位素訊號峰群中的間隔大小就可以知道了；如果間隔 大小為1.0，則是帶1個正電荷；如果間隔大小為0.5，則是帶2個正電荷；如果間隔大小為 0.33，則是帶3個正電荷；如果間隔大小為0.25，則是帶4個正電荷；依此可類推。此種方式 一般應用於分子量較小的胜肽，因為胜肽的氨基酸數少，帶正電荷數目也小，並且一般的質 譜儀就可以將同位素訊號峰群解析的很好，據以計算訊號峰所對應之帶正電荷數目不是問 題。相反的，分子量大於10,000 Da以上的蛋白質，帶正電荷數目大，除非利用超高解析度 的質譜儀，一般的質譜儀無法將同位素訊號峰群解析的很好，多數情況下利用第一種方式計 算訊號峰所對應的電荷數目。

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NanoSpray Interface for Nano LC/MS

52

53 54

Before 1980, protein identification only for Edman Protein (peptide) is macro-molecule, difficult to ionization After 1990, (MALDI & ESI) combine with bio-informatic analysis Human genome project: 30,000 gene → 90,000 protein → PTM

→ >400,000 protein

Edman sequence vs. MS

Edman : 2-3 amino acids / hr MS + computer analysis: > 100 fold Sensitivity: MS better

55

與以往的離子化方式不同的，上述這兩類技術能 (MALDI & ESI)使得蛋白質轉變為氣態離子而不會 改變其結構與形體，所以能快速準確地測定生化大 分子的分子量；再結合各種新的質譜分析技術，便 可以為蛋白質研究開闢了新的道路。生化大分子多 為極性、難揮發化合物，不易氣化，用傳統質譜無 法測定。但隨著新的離子化技術的廣泛應用，現已 能高效地離子化完整或片斷的生化大分子聚合物，

從而進行質譜測定。由於這兩種離子化方式擴大了 質譜儀的應用範圍，故得到2002年的諾貝爾化學獎 的肯定。

56

MALDI VS. ESI

Similarity

1. 2002 Nobel Prize

2. Soft ionization (keep peptide intact) 3. Molecular weight analysis good 4. Protein identification ok

5. Sensitivity (< pmol)

6. Positive charge peptide (or protein) 7. > 10000 Da → can not alanysis

57

General sample handling

58

MALDI/ESI comparison MALDI/ESI comparison

Feature MALDI ESI

Sample Solid Liquid

Ion charge state Lower charge states

Higher charge states

High throughput Easier More difficult

Mass limit > 1,000,000 Da > 1,000,000 Da Detection limit Attomole (10^-18

mol)

Attomole (10^-18mol)

Salt (etc.) tolerance

Good Bad

Analysis of mixtures

Easier More difficult

On-line separations

More difficult Easier

59

超高速

高速

低速 極豐富

多量 中量

蛋白質體分析邏輯 蛋白質體分析邏輯

樣品分析量

資訊獲得量

MALDI-TOF MS MALDI-TOF MS/MS nanoESI-MS/MS

• MALDI-TOF 快速篩選研究目標蛋白，減少 ESI-LC-MS/MS 工作負擔。

• ESI-LC-MS/MS 細部分析微量蛋白，研究 Post-Translational Modification.

• 建立 Bioinformatics 系統，分析大量結果，修正研究結論。

MALDI-TOF MS ESI LC/MSⁿMS

Bruker Daltonics, Taiwan Office ⁶⁰

MALDI VS. ESI

MALDI ESI

Application Protein identification Nature compound, metabolite, organic chemistry, protein

analysis Salt impurities

tolerance

Tolerance bad better Sample condition Solid liquid

Ionization impulse continuous Protein mixture

identification

bad good

Combine with LC NO YES

Analysis time short long

Data information less much

charge +1 +1.2.3…

Content analysis NO YES

61

Ionization Methods for Proteomics:

ESI vs. MALDI

• ESI advantages

– Direct coupling of LCto MS - Sophisticated workflows – Established software

packages

– Accepted MS/MS ionization mode

• ESI disadvantages – Limited timefor MS/MS

(limited depth of analysis) – Lack of ability to perform result dependent analysis – Miss high MWpeptides

• MALDI Advantages – Sample in solidstate – Not time-limited for MS/MS – Sophisticated workflows – Fast – lots of MS and

MS/MS – Data dependent

acquisition stop criteria

• MALDI disadvantages – Offline coupling of LC – Miss low MWpeptides

62

63

Mass analyzer (質量分析器)

TOF (Time of flight) 飛行時間 Quadrupole 四極棒式

Ion-trap 離子阱

Magnetic field defection 磁場反射式 (1980)

Fourier transform ion cyclotron resonance (FT-ICR)傅立葉轉換 Usually to proteomic study

64

Ion-trap 離子阱

離子阱式質譜儀( ion trap mass spectrometers）

離子阱式質譜儀基本原理則是利用一個三維的電場將帶電離子 困住，再改變電場讓不同的質荷比的離子一個個的自離子阱中 射出，再來偵測訊號。

65 66

Ion-trap (flash movie)

Step 1: enter ion-trap Step 2: ion trapping Step 3: capture (捕獲) Step 4: collision (碰撞) Step 5: detection

67

Quadrupole (Q) Mass Analyzer

Uses a combination of RF and DC voltages to operate as a mass filter.

•Has four parallel metal rods.

•Lets one mass pass through at a time.

•Can scan through all masses or sit at one fixed mass.

四極柱式質譜儀（Quadrupole Mass Spectrometer)

四極柱式質譜儀基本原理是利用一固定的施加電位下，使只有某一個質荷比

（m/e）的離子呈穩定振動並通過四極圓柱，到達偵測器而被偵測到，其他質荷 比之離子則因軌道振幅越來越大而撞擊到四極柱上而被中和。

RF: radiofrequence voltage

68

mass scanning mode

m3 m1

m4 ^m2

m3

m1

m4

m2

single mass transmission mode

m2 m2 m2 m2

m3

m1

m4

m2

Quadrupoles have variable ion transmission modes

69

Quadrupole Analyser

+ +

- -

ion path 4 parallel rods with a DC voltage &

a superimposed radiofrequency (rf) potential

rf for each pair of rods is same, but differ in phase by 180°

rf field istunable - quadrupole acts as a filter to allow only those ions with a specific m/z ratio to pass through

70

m/z = 10 m/z = 100m/z = 1000

To detector

How the quadrupole acts as a filter

set rf to allow only a subset of ions to pass through

71

Quadrupole

Step I: full scan Step II: production ion scan

72

Step III: precursor ion scan

73

ESI + quadropole

74

Magnetic Mass Analyzer

ion stream is subjected to a magnetic field; ions are deflected to different extents based on m / z differences

S N

ions with low m/z deflected the most 磁場反射式

75

FT-ICR.

Also uses E & B but v. low K.E. Ions become trapped in magnetic fields . Resolution in the millions is possible with ppm accuracy, but $$$.

傅立葉轉換

76

Reflectrontime-of-flight mass spectrometer Lineartime-of-flight mass spectrometer

Time of flight (TOF) Mass Analyzer 飛行時間式質譜儀（Time of flight，TOF)

其測定的基本原理為，將帶電離子在一個真空室中加速射出，

然後測量其落地前所需之飛行時間。它們達到偵測器的先後與 其質量以及電荷有關，越輕而且電荷越高者，越快抵達。

Time of Flight (TOF)

A footrace from the site of ionization to the detector, under the influence of an electric field

Highly charged, low molecular weight ions (low m/z) reach the detector first

77

Time-of-flight (TOF) Mass Analyzer

+

+

+

+

Source Drift region (flight tube)

detector

V

•Ions are formed in pulses.

•The drift region is field free.

•Measures the time for ions to reach the detector.

•Small ions reach the detector before large ones.

78

++ +

+ +

+++ +

+

pulsed UV or IR laser (3-4 ns)

detector

vacuum

strong electric field

Time Of Flight tube peptide mixture

embedded in light absorbing chemicals (matrix)

cloud of protonated peptide molecules Vacc

79 Linear Time Of Flight tube

Reflector Time Of Flight tube

detector

reflector ion source

ion source

detector

time of flight

time of flight

80

Time-of-Flight Mass Spectrometry

In time-of-flight (TOF) instruments, positive (+) ions are produced periodically (週期性) by bombardment (轟炸) of the sample with brief pulses of electrons, secondary ions, or in this case laser-generated photons.

These laser pulses typically have a frequency of 10 to 50 kHz and a lifetime of 0.25 μs.

The ions produced by the laser are then accelerated by an electric field pulse of 10³to 10⁴V that has the same frequency as, but lags behind, the ionization pulse The accelerated particles then pass into a field-free drift

tube. The drift tube’s length can range from 0.5 - 3.0 meters.

81

Post Source Decay

• Thus, it came rather as a surprise that roughly the same kind of information,as in the ‘protein ladder’ sequencing, can be obtained as a by-product in reflectron TOF spectrometers

– Simply by the analysis of the abundant post source decay (PSD) fragments

– These fragments are formed in the field-free drift region after MALDI

• Need a reflectron TOF spectrometer and a precursor ion selector

82

Reflectron & Post Source Decay

• Linear TOF machines- fragmented in flight by only mass.

Ensemble of decay products travel with parent ion.

• If, however, a retarding field is placed in front of the detector, the extent of fragmentation is easily measured

• The measuring principle uses the fact that the kinetic energy of a fragment ion is proportional to its fractional mass. Thus, in the retarding field of the reflectron, such fragments will turn around earlier than their respective precursors and will arrive at the detector sooner.

83

Post Source Decay (PSD) fragment ion mass analysis

z

evaluation of post source decay following MALDI

R. Kaufmann, J. Biotechnol. 4, 160 (1995). 84

QSTAR

QSTAR

ESI QQ TOF or MALDI QQ TOF ESI QQ TOF or MALDI QQ TOF

Q1

Ion Mirror (reflector) Effective Flight Path = 2.5 m

Q2 Q0

Sample

85

Quadrupole + refrectron TOF

86

How do mass spectrometers get their names?

Types of ion sources:

• Electrospray (ESI)

• Matrix Assisted Laser Desorption Ionization (MALDI) Types of mass analyzers:

• Quadrupole (Quad, Q)

• Ion Trap

• Time-of-Flight (TOF)

-Either source type can work with either analyzer type: “MALDI- TOF,” “ESI-Quad.”

-Analyzers can be combined to create “hybrid” instruments.

ESI-QQQ, MALDI QQ TOF, Q Trap

87

Mass Spectrometer

Tandem Mass Spectrometer (串聯式質譜)

88

MS vs. Tandem MS

89

Collision-induced dissociation (CID)

In 碰撞誘起解離

(CID)

A B

a1 a2 b1 b2

若利用單一質譜測定分子離子的荷質比，會無法分辨組成相同但排列序列 不同的分子。為提高分子鑑定率，於是發展出串聯質譜（Tandem mass spectrometers）分析。所謂的串聯質譜是在第一次質譜得到分子離子後，

選取擁有特定荷質比的離子作為母離子（parent ion），與惰性氣體碰撞，

產生碰撞誘導解離（Collision-Induced Dissociation，CID），形成一系列 子離子（daughter ion），這時排列序列不同會產生不同的子離子系列，如 此一來，便可分辨組成相同但排列序列不同擁有同分子。結合四極柱式與 飛行時間的Q-TOF、反射式TOF（Reflection TOF）及Ion Trap都可進行串 聯質譜分析。

90

CID induced Peptide Fragmentation

• Peptides tend to fragment along the backbone.

• Fragments can also loose neutral chemical groups like NH

and H

O.

H...-HN-CH-CO . . . NH-CH-CO-NH-CH-CO-…OH

R_i-1 R_i R_i+1

H⁺

Prefix Fragment Suffix Fragment

Collision Induced Dissociation

91

-HN--CH--CO--NH--CH--CO--NH-

R

CH-R’

c_i

z_n-i

R”

d_i+1 v_n-i w_n-i

low energy

high energy

Cleavages Observed in MS/MS of Peptides

a_i

x_n-i

b_i

y_n-i

92

E G S F F G E E N P N V A R

Peptide Fragmentation

175.10 246.14 345.21 459.25 556.30 670.35 799.39 928.43 985.45 1132.52 1279.59 1366.62 1423.64 1552.69

=>

=

=

=

E=Glu G=Gly S=Ser F=Phe N=Asn P=Pro V=Val A=Ala R=Arg

93

What is MS/MS?

MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.

Ion

source MS-1 MS-2

Mixture of ions

Single ion

Fragments

94

Tandem Mass Spectrometry

Tandem Mass Spectrometry --MS/MSMS/MS

MS1 Q2 MS2

MASS FILTER Collision Cell MASS FILTER

PRECURSOR ION SEPARATION

NEUTRAL GAS COLLISIONS

PRODUCT ION SEPARATION ION

SOURCE

MS Spectrum

MS/MS Spectrum

ION DETECTOR

• Ions are produced from all peptides in the sample in ion source

• Only one type of peptide passes through MS1

• This peptide collides with gas molecules in Q2 and fragments

• The fragments from this peptide are analyzed in MS2

• This MS/MS spectrum provides the amino acid sequence of this peptide

95

Electrospray Ionization Source

MS-1

ESI-Tandem MS

MS-2

Collision Cell Detector

ES Source

MS-1 MS-2

Collision Cell

Detector

Input: peptides from enzymatic digest

Select for a particular ion (peptide) P1

P2

P3

P4

P5

He gas

F₁F₂ F₃F₄ F₅

Output: fragments from daughter ions

96

What is MS/MS?

MS/MS

+ +

+ +

+ 1 peptide

selected for MS/MS

The masses of all the pieces give an MS/MS spectrum Peptide

mixture

Have only masses to start

97

Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle

+ +

+ +

+

Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together

Inlet Ion source

Mass

Analyzer Detector Data System High Vacuum System

Microchannel Plate Electron Multiplier Hybrid with photomultiplier

Detector

99 +

e^- primary ion

e^- e^-e^- L

D

-1000V

-100V

L >> D

Ions are detected with a microchannel plate

Inlet Ion source

Mass

Analyzer Detector Data System High Vacuum System

PC

Sun SPARK Station DEC Station

Data System

101

The mass spectrum shows the results

Relative Abundance

Mass (m/z)

0 10000 20000 30000 40000

50000 100000 150000 200000

MH⁺

(M+2H)²⁺

(M+3H)³⁺

MALDI TOF spectrum of IgG

102

ESI Spectrum of Trypsinogen (MW 23983)

1599.8

1499.9 1714.1

1845.9 1411.9

1999.6 2181.6

M + 15 H⁺

M + 13 H⁺ M + 14 H⁺ M + 16 H⁺

m/z Mass-to-charge ratio

103

Isotopes

+Most elements have more than one stable isotope.

For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.

+Why do we care?

Mass spectrometers can “see” isotope peaks if their resolution is high enough.

If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved.

104

Element Mass Abundance

H 1.0078

2.0141

99.985%

0.015

C 12.0000

13.0034

98.89 1.11

N 14.0031

15.0001

99.64 0.36

O 15.9949

16.9991 17.9992

99.76 0.04 0.20

Stable isotopes of most abundant elements of peptides

105

1981.84 1982.84

1983.84

Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)

No ¹³C atoms (all ¹²C)

One¹³C atom

Two¹³C atoms

“Monoisotopic mass”

106

m/z 4360.45

4361.45

Isotope pattern for a larger peptide (207 C-atoms)

107

Average mass

Average mass corresponds to the centroid of the unresolved peak cluster

When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.

108

6130 6140 6150 6160 6170

Poorer resolution Better

resolution

What if the resolution is not so good?

At lower resolution, the mass measured is the average mass.

Mass

15.01500 15.01820 15.02140 15.02460 15.02780 15.03100 109 Mass (m/z)

100

0 10 20 30 40 50 60 70 80 90 100

% Intensity

ISO:CH3 15.0229

M

FWHM = ΔM

R = M/ΔM

How is mass resolution calculated?