Name: how esi turns biomolecules into ions
Uploaded: 2024-12-05T18:32:21.000Z
Duration: 1 min 12 s
Channel: JoVE

タンパク質や核酸などの生体分子は、高分子量で揮発性の低い化合物です。従来の電子インパクトイオン化質量分析は、このような分子に対して、深刻な分子断片化などの課題に直面していました。
エレクトロスプレーイオン化は、このような生体分子の質量分析のために非ラジカル分子イオンを生成する代替の「ソフト」イオン化法です。
この方法では、サンプルをメタノール、メタノール水、アセトニトリルなどの極性溶媒と混合します(塩を添加する、または添加しない)。
混合物は、高電圧キャピラリーを介して真空チャンバーに噴霧されます。帯電した液滴の微細なスプレーから溶媒が急速に蒸発すると、最終的に帯電したガス状のサンプル分子が得られます。
この荷電したサンプル分子は、サンプル分子量と関連イオンの質量の合計としてマススペクトルで検出されます。
例えば、イオン溶液中にナトリウムイオンが存在する場合、スペクトルは通常、M+23のピークを特徴とします。

エレクトロスプレーイオン化 (esi) 質量分析法

Electrospray Ionization (ESI) Mass Spectrometry

分子量の大きい生体分子は不揮発性化合物であり、従来の電子衝撃イオン化法による質量分析ではイオン化または気化する前に分解する可能性があります。したがって、エレクトロスプレーイオン化 (ESI) は、急速なフラグメンテーションを回避し、生体分子全体の質量信号を記録できるため、生体分子の気化およびイオン化に好まれる方法です。

ESI は、電気エネルギーを利用してサンプルの液相から気相にイオンを移動させます。分析物生体分子は、イオン化源として機能する非分子化合物であるイオン液体と事前に混合されます。この溶液は高電圧キャピラリーを通して噴霧され、微細なエアロゾルを生成します。キャピラリーと分析チャンバーの入口の間に適用される電圧差により、帯電した液滴が分析チャンバーに向かって移動します。この動きの間にエアロゾルのイオン液体が蒸発し、さまざまな程度にプロトン化された、またはイオン化された分析物分子が残ります。これらの荷電種は、分析物分子の分子量と、分析物分子に関連するイオン液体のイオンの分子量の合計に相当する m/z 値で質量スペクトルで検出されます。

質量信号の位置は、イオン液体中に存在する陽イオンの種類によって異なります。たとえば、イオン液体中にナトリウムイオンが存在すると、質量スペクトルで (M+23) ピークが生じますが、プロトンが存在すると (M+1) ピークが生じます。

エレクトロスプレーイオン化 (ESI) 質量分析法

高分子量の生体分子は不揮発性化合物であり、従来の質量分析ではイオン化または気化する前に分解する可能性があります...

electrospray-ionization-esi-mass-spectrometry

Education

JoVE Core

Analytical Chemistry

分析化学

Unit 1

ユニット1

Mass Spectrometry Fragmentation Methods

質量分析におけるフラグメンテーション法

KEY TERMS AND CONCEPTS

主要用語と概念

how esi turns biomolecules into ions

Electrospray ionization, or ESI, is a mass spectrometry method used to detect biomolecules that are too large or nonvolatile for conventional electron impact ionization. These molecules may break down before they can ionize or evaporate. ESI helps prevent rapid fragmentation and lets scientists record a mass signal for the whole biomolecule.

ESI uses electrical energy to move ions from the liquid phase into the gas phase. The analyte biomolecule is mixed with an ionic liquid, which is a nonmolecular compound that acts as the ionization source. The solution then passes through a high-voltage capillary and forms a fine aerosol.

A voltage difference between the capillary and the entrance to the analysis chamber pulls the charged droplets toward the chamber. As the droplets travel, the ionic liquid evaporates. This process leaves behind a protonated or otherwise ionized analyte molecule.

The charged species are measured in the mass spectrum by their m/z value, which reflects the analyte’s molecular mass plus the mass of the ions from the ionic liquid attached to it. The peak position depends on which cations are present in the ionic liquid. For example, sodium ions produce an (M+23) peak, while a proton produces an (M+1) peak.

How ESI Turns Biomolecules Into Ions

mass spectrometry: long-chain alkane fragmentation

mass-spectrometry-long-chain-alkane-fragmentation

Mass Spectra of Linear Alkane Chains

Mass spectra of linear alkanes show a clear fragmentation pattern. The molecular ion often breaks at a carbon-carbon bond away from the end of the chain. This cleavage forms a stable carbocation and a stable radical, so the middle of the mass-to-charge plot usually has the strongest signals while the ends are weaker. As each bond breaks, a methyl group is lost and the main peaks in the spectrum appear 14 u apart. Smaller peaks near each main signal can also appear. These come from secondary...

mass spectrometry: branched alkane fragmentation

mass-spectrometry-branched-alkane-fragmentation

Branched Alkanes in Mass Spectra

Branched alkanes in mass spectrometry fragment quickly because branching can create stable carbocations. When cleavage happens at a branching point, a secondary or tertiary carbocation may form. That stability helps explain why the molecular ion peak is often very weak or missing compared with a linear alkane. The lesson uses 2-methylbutane, neopentane, and n-pentane to show how this pattern appears in mass spectra. 2-methylbutane and neopentane break apart to form secondary and tertiary...

mass spectrometry: cycloalkane fragmentation

mass-spectrometry-cycloalkane-fragmentation

Cycloalkane Ions and Mass Peaks

Cycloalkane ions in mass spectrometry often give strong peaks because the ring structure helps stabilize the molecular ion. Compared with linear or branched alkanes, this added stability changes how the molecule breaks apart. As a result, cycloalkanes can produce prominent signals in the mass spectrum. Cyclohexane is a clear example. Its molecular ion has an m/z of 84, and this peak is often stronger than the signal from hexane, a linear alkane. The closed ring helps hold the ion together,...

mass spectrometry: alkene fragmentation

Alkene Ion Fragmentation Patterns

Alkene ion fragmentation patterns in mass spectrometry begin when the molecule loses one electron from the unsaturated π bond. This ionization forms a stable molecular ion. The ion can then break apart by three main reaction pathways. The most common pathway is cleavage at the allylic position, which is the carbon next to the double bond. This produces an allylic carbocation that is stabilized by resonance. In terminal alkenes, this fragment appears at a mass-to-charge ratio of 41. In internal...

mass spectrometry: cycloalkene fragmentation

mass-spectrometry-cycloalkene-fragmentation

Cycloalkene Side-Chain Cleavage in Mass Spectra

Cycloalkenes can break apart in a mass spectrometer through retro-Diels–Alder fragmentation. In this process, two carbon-carbon bonds in the ring split, and ethene is released. The remaining fragment is a dienyl radical cation that is 28 u less than the molecular ion. This pathway is similar to the fragmentation of cycloalkanes that also releases ethene. The difference is the type of charged fragment that remains. Cycloalkanes form an alkyl species, while cycloalkenes form a dienyl species.

mass spectrometry: alkyne fragmentation

Alkyne Mass Spectra and Key Peaks

Alkyne mass spectra often show strong peaks from two main fragmentation paths. One path breaks the carbon-carbon bond between the alpha and beta carbons of the alkyne bond. This produces a 3-propynyl cation, also called a propargyl cation. In terminal alkynes, this is the only fragmentation path that gives the 3-propynyl cation. The unsubstituted 3-propynyl cation appears at a mass-to-charge ratio of 39. In internal alkynes, the 3-propynyl cation is substituted. For example, 2-pentyne forms a...

mass spectrometry: alcohol fragmentation

Alcohol Fragmentation Patterns in Mass Spectra

Alcohols fragment in mass spectrometry in predictable ways. When an alcohol, written as R-OH, ionizes, it can lose one non-bonded electron from the oxygen atom. That forms a molecular ion, but the molecular ion peak is often weak or may not appear because alcohols break apart quickly. Two main fragmentation routes are important for alcohols: β-cleavage and dehydration. In β-cleavage, the bond at the β-position next to the hydroxyl group breaks. This gives a resonance-stabilized cation and a...

mass spectrometry: aromatic compound fragmentation

mass-spectrometry-aromatic-compound-fragmentation

Mass Spectra of Benzene and Toluene

Mass spectra of aromatic compounds show a strong molecular ion peak. In ionization, the molecule becomes a molecular ion, which appears as a prominent peak in the spectrum. Benzene gives a molecular ion at m/z 78, and toluene gives one at m/z 92. Benzene’s molecular ion is especially stable. It does not fragment readily because breaking the aromatic benzene ring requires a large amount of energy. Alkyl-substituted benzenes behave differently in mass spectrometry. Toluene can lose a hydrogen...

mass spectrometry: amine fragmentation

Amines in Mass Spectra: Nitrogen Rule

Amines can be identified in mass spectrometry by their fragmentation patterns and by the nitrogen rule. The molecular ions of amines often break apart through β-cleavage, which cuts a carbon-carbon bond next to the nitrogen. This process forms an alkyl radical and a resonance-stabilized nitrogen-containing cation. The nitrogen rule helps predict the mass of the molecular ion. A compound with one nitrogen atom, or any odd number of nitrogen atoms, gives a molecular ion with an odd molecular...

mass spectrometry: alkyl halide fragmentation

mass-spectrometry-alkyl-halide-fragmentation

Mass Spectra of Alkyl Halides

Mass spectra of alkyl halides show clear isotope patterns from the halogen atom. Chlorine has two common isotopes, 35Cl and 37Cl, in a 3:1 ratio. Bromine has two common isotopes, 79Br and 81Br, in a 1:1 ratio. These isotope ratios create two molecular ion peaks, [M] and [M + 2], with relative heights that match the halide abundance. This pattern is easy to see in examples such as 2-chloropropane and 1-bromopropane. 2-Chloropropane gives peaks in a 3:1 ratio. 1-Bromopropane gives peaks in a 1:1...

mass spectrometry: aldehyde and ketone fragmentation

mass-spectrometry-aldehyde-and-ketone-fragmentation

Carbonyl Fragment Patterns in Mass Spectra

Aliphatic aldehydes and ketones break apart in mass spectrometry through a few key carbonyl fragment patterns. The main pathways are alpha-cleavage, inductive cleavage, and the McLafferty rearrangement. These patterns help chemists read the mass spectrum and spot a carbonyl group. Alpha-cleavage is a common break next to the carbonyl group. The bond beside the carbonyl splits, and this forms an alkyl radical and an acylium cation. The acylium cation is often seen in the spectrum because it is...

mass spectrometry: carboxylic acid, ester, and amide fragmentation

mass-spectrometry-carboxylic-acid-ester-and-amide-fragmentation

Mass Spectra of Carboxylic Compounds

Mass spectra of carboxylic acids, esters, and amides often show two key fragmentation routes: β-cleavage and McLafferty rearrangement. In β-cleavage, the carbon-carbon bond at the β-position next to the carboxylic group breaks. This produces a neutral radical and a cation. Long-chain compounds can also fragment by McLafferty rearrangement if they have a hydrogen on the γ-carbon. The γ-carbon is the third carbon away from the carboxylic group. This rearrangement gives a radical cation and a...

chemical ionization (ci) mass spectrometry

Using Reagent Gas in CI Mass Spectrometry

Chemical ionization mass spectrometry uses a reagent gas to help detect molecules that break apart too quickly in electron impact ionization. It is a milder ionization method that can extend the life of the ionized analyte, which makes molecular identification easier. In chemical ionization, the sample is mixed with an excess reagent gas. This setup makes the electron impact occur mainly on the reagent gas instead of on the analyte. The charged species formed from the reagent then protonates...

electrospray ionization (esi) mass spectrometry

Electrospray ionization, or ESI, is a mass spectrometry method used to detect biomolecules that are too large or nonvolatile for conventional electron impact ionization. These molecules may break down before they can ionize or evaporate. ESI helps prevent rapid fragmentation and lets scientists record a mass signal for the whole biomolecule. ESI uses electrical energy to move ions from the liquid phase into the gas phase. The analyte biomolecule is mixed with an ionic liquid, which is a...

matrix-assisted laser desorption ionization (maldi)

matrix-assisted-laser-desorption-ionization-maldi

How MALDI Turns Samples Into Ions

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry technique that turns biomolecules into ions for analysis. It is used to identify and characterize proteins, peptides, nucleic acids, and carbohydrates. Because of this, it is common in biological and medical research, as well as pharmacology and biochemistry. In MALDI, the analyte of interest is mixed with a matrix material. The matrix is usually an organic compound, such as a small organic acid or a crystalline...

key terms and concepts

Learn how electrospray ionization moves biomolecules from liquid to gas, forms charged droplets, and creates peaks such as M+23 and M+1 in mass spectrometry.

How ESI Turns Biomolecules Into Ions | JoVE K12

JoVE Core 分析化学 は、複雑な化学原理をアニメーションで視覚化することで、SI 単位から NMR 分光法を含むトピックをよりわかりやすくします。実験を撮影した実写動画では、これらの概念が世界中の研究所でどのように応用されているかを取り上げています。

How ESI Turns Biomolecules Into Ions

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