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

Биомолекулы, такие как белки и нуклеиновые кислоты, представляют собой соединения с низкой летучестью и высокой молекулярной массой. Традиционная ионизационная масс-спектрометрия с электронным ударом сталкивается с проблемами, связанными с такими молекулами, включая сильную молекулярную фрагментацию.
Ионизация электрораспылением является альтернативным методом «мягкой» ионизации, который генерирует нерадикальные молекулярные ионы для масс-спектрометрии таких биомолекул.
В этом способе образец смешивают с полярным растворителем, таким как метанол, метанол-вода или ацетонитрил, с добавлением солей или без них.
Смесь распыляется через высоковольтный капилляр в вакуумную камеру. Быстрое испарение растворителя из мелкодисперсного распыления заряженных капель в конечном итоге приводит к образованию заряженной газообразной молекулы образца.
Эта заряженная молекула образца обнаруживается в масс-спектре как сумма молекулярной массы образца и массы связанных ионов.
Например, если в ионном растворе присутствуют ионы натрия, то в спектре обычно присутствует пик M+23.

Масс-спектрометрия с ионизацией электрораспылением (esi)

Electrospray Ionization (ESI) Mass Spectrometry

Биомолекулы с более высокой молекулярной массой являются нелетучими соединениями, которые могут разлагаться до ионизации или испарения во время массового анализа с использованием обычных методов ионизации электронным ударом. Соответственно, ионизация электрораспылением (ESI) является предпочтительным методом испарения и ионизации биомолекул, поскольку она позволяет избежать быстрой фрагментации и позволяет регистрировать массовые сигналы для всей биомолекулы.

ESI использует электрическую энергию для переноса ионов из жидкой фазы образца в газообразную фазу. Биомолекула аналита предварительно смешивается с ионной жидкостью — немолекулярным соединением, служащим источником ионизации. Раствор распыляется через капилляр высокого напряжения для получения мелкодисперсного аэрозоля. Разность напряжений, приложенная между капилляром и входом в аналитическую камеру, вызывает движение заряженных капель по направлению к аналитической камере. Ионная жидкость из аэрозоля испаряется во время этого движения, оставляя после себя переменно протонированную или ионизированную молекулу аналита. Эти заряженные виды обнаруживаются в масс-спектре при значении m/z, эквивалентном сумме молекулярной массы молекулы аналита и молекулярной массы ионов из ионной жидкости, связанной с молекулой аналита.

Положение масс-сигналов меняется в зависимости от типа катионов, присутствующих в ионной жидкости. Например, присутствие ионов натрия в ионной жидкости приводит к пику (M+23) в масс-спектре, тогда как присутствие протона приводит к пику (M+1).

Масс-спектрометрия с ионизацией электрораспылением (ESI)

Биомолекулы с более высокой молекулярной массой - это нелетучие соединения, которые могут разлагаться перед ионизацией или испарением во время массового анализа с помощью обычных ...

electrospray-ionization-esi-mass-spectrometry

Education

JoVE Core

Analytical Chemistry

Аналитическая химия

Unit 1

Раздел 1

Mass Spectrometry Fragmentation Methods

Методы фрагментации в масс-спектрометрии

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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

analytical chemistry

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How ESI Turns Biomolecules Into Ions

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