Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5647_Библиотеки_им_академика_М_И_Перельмана.pdf
9.3 Instrumentation 371
FAB was one of the early soft ionization techniques developed to ionize large, polar,
and thermally sensitive molecules that could not be analyzed effectively using
traditional ionization methods such as electron ionization (EI).
9.3.2.5.1 Principle of FAB
In FAB, the sample is dissolved in a viscous matrix and bombarded by a beam of
high-energy neutral atoms, typically argon or xenon atoms. This bombardment
transfers energy to the sample, causing desorption and ionization of the analyte
molecules, which are then introduced into the mass spectrometer for analysis. FAB
is a soft ionization technique, producing predominantly molecular ions (M
+
) with
minimal fragmentation, which makes it useful for determining molecular weights of
sensitive compounds.
9.3.2.5.2 Key Features of FAB
• Soft ionization: FAB results in the production of molecular ions with minimal
fragmentation, allowing the determination of molecular weights without significant structural breakdo wn.
• Viscous matrix: The sample is mixed with a viscous matrix (e.g., glycerol or
thioglycerol), which helps protect the sample from excessive fragmentation and
assists in the transfer of energy from the atom beam.
• Neutral atom beam: A beam of fast-moving neutral atoms (commonly argon or
xenon) is directed at the sample, causing desorption and ionization of the analyte
molecules.
• Applicable to nonvolatile compounds: FAB is particularly useful for the analysis
of polar, nonvolatile, and thermally labile compounds that are not suitable for
traditional ionization methods such as EI or CI.
9.3.2.5.3 Process of FAB
1. Sample preparation: The analyte is dissolved in a viscous matrix that can
withstand the bombardment process. Common matrices include glycerol, which
helps in ion formation and stabilization.
2. Atom bombardment: A beam of high-energy neutral atoms (e.g., argon or xenon)
is directed at the sample matrix. The collisions between the neutral atoms and the
sample cause the analyte molecules to desorb and ionize.
3. Ion formation: The energy transfer from the atom beam results in the formation of
molecular ions (M
([M + H]
+
or [M - H]-). Fragmentation is typically minimal.
+
) and sometimes protonated or deprotonated species
4. Mass analysis: The ionized molecules are then analyzed by the mass spectrome-
ter to determine their mass-to-charge ratio (m/z) and obtain information about
their molecular weight and structure.
9.3.2.5.4 Advantages of FAB
• Minimal fragmentation: FAB
is a soft ionization method that produces molecular
ions with minimal fragmentation, making it ideal for determining the molecular
weight of sensitive compounds.
372 9 Comprehensive Insights into Mass Spectrometry
• Analysis of polar and nonvolatile compounds: FAB is especially useful for
analyzing polar, nonvolatile, and thermally sensitive compounds such as
peptides, nucleotides, lipids, and certain synthetic polymers.
• Applicable to biological molecules: FAB is suitable for analyzing large
biomolecules, such as peptides and
oligosaccharides,
which are challenging to
analyze using tradi tional ionization methods.
9.3.2.5.5 Limitations of FAB
• Matrix interference: The matrix used in FAB can sometimes produce background
ions or interfere with the ionization of the analyte, making it challenging to
analyze certain samples.
• Limited mas s range: FAB is less effective for very h igh molecular weight
compounds compared to more modern techniques such as electrospray ionization
(ESI) or matrix-assisted laser desorption ionization (MALDI).
• Low sensitivity: FAB generally has lower sensitivity compared to newer ioniza-
tion techniques, making it less suitable for trace-level analysis.
9.3.2.5.6 Applications of FAB
• Peptide and protein analysis: FAB has been historically used for the analysis of
peptides and small proteins, providing molecular weight information without
significant fragmentation.
• Nucleotide and oligosaccharide analysis: FAB
is useful
for the analysis of
nucleotides and oligosaccharides, especially when determining molecular
weights and simple structural features.
• Lipid and organic molecule analysis: FAB is also applied to the analysis of polar
organic molecules, such as lipids and certain synthetic polymers, which are
difficult to ionize using other methods.
9.3.2.6 Electron Capture Dissociation (ECD) and Electron Transfer
Dissociation (ETD)
Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are
both fragmentation techniques used in mass spectrometry, primarily for the analysis
of large biomolecules such as proteins and peptides. These methods are designed to
preserve posttranslational modifications (PTMs) such as phosphorylation and glycosylation, making them particularly useful for proteomics research. ECD and ETD are
both considered “radical-based dissociation” methods that cleave the peptide backbone while preserving the integrity of labile side chains and modifications.
.1 E
9.3.2.6
lectron Capture Dissociation
ECD is a technique where a multiply protonated peptide or protein ion (typically
generated via electrospray ionization or another ionization source) captures a
low-energy electron, leading to the cleavage of the peptide backbone at the N–Cα
bond. This results in the production of fragment ions, particularly c- and z-ions,
without disrupting side-chain modifications, allowing the analysis of PTMs.
9.3 Instrumentation 373
9.3.2.6.1.1 Principle of ECD
• A protonated analyte (typically a peptide or protein) interacts with low-energy
electrons.
• The electron is captured by the analyte, which leads to radical-induced cleavage
of the peptide backbone.
• Fragmentation predominantly occurs at the N–Cα bond, resulting in the produc-
tion of c- and z-type fragment ions.
9.3.2.6.1.2 Key Features of ECD
• Selective cleavage: ECD cleaves the N–Cα bond, resulting in c- and z-ions, which
provide detailed information about the peptide backbone.
• Minimal fragmentation of side chains and PTMs: Labile posttranslational
modifications (e.g., phosphorylation, glycosylation) remain intact, making ECD
ideal for studying modified peptides and proteins.
• Soft fragmentation method: ECD induces fragmentation via radical chemistry,
preserving the overall integrity of the molecule and generating informative
fragments.
9.3.2.6.1.3 Advantages of ECD
• Retention of PTMs: ECD preserves posttranslational modifications such as phos-
phorylation and glycosylation, making it highly useful for studying protein
modifications.
• Complem entary to other fragmentation methods: ECD can be used in combina-
tion with other fragmentation techniques (such as CID and HCD) to provide
complementary fragmentation data.
• Useful for large biomolecules: ECD is highly effective for analyzing large,
multiply charged biomolecules such as proteins and complex peptides.
9.3.2.6.1.4 Applications of ECD
• Proteomics: ECD is widely used in proteomic studies to sequence peptides and
proteins while preserving posttranslational modifications.
• Structura l analysis of proteins: ECD provides detailed information on protein
backbone structure while retaining labile modifications, making it useful for
characterizing protein structures and modifications.
• Study of disulfide
bonds: ECD
is effective at cleaving disulfide bonds, providing
information on protein folding and structure.
9.3.2.6
ETD is
lectron Transfer Dissociation
.2 E
a fragmentation method similar to ECD but uses a different mechanism to
induce fragmentation. In ETD, an anionic reagent (usually a radical anion such as
anthracene or fluoranthene) transfers an electron to the multiply charged cationic
analyte (e.g., a protonated peptide), leading to fragmentation of the peptide backbone
at the N–Cα bond. Like ECD, ETD preserves labile modifications, such as phosphorylation and glycosylation.
374 9 Comprehensive Insights into Mass Spectrometry
9.3.2.6.2.1 Principle of ETD
• An electron-rich reagent anion transfers an electron to a multiply protonated
peptide or protein.
• This electron transfer induces fragmentation of the peptide backbone, producing
c- and z-type ions.
• Like ECD, the fragmentation preserves labile posttranslational modifications.
9.3.2.6.2.2 Key Features of ETD
• Electron transfer: Unlike ECD, which directly captures electrons, ETD uses an
electron-donating anion to transfer an electron to the analyte, causing peptide
backbone fragmentation.
• Fragmentation of N–Cα bond: ETD cleaves the N–Cα bond, producing c- and z-
ions, providing sequence information without disrupting side-chain
modifications.
• Similar to ECD: ETD produces results comparable to ECD, preserving PTMs and
labile structures.
9.3.2.6.2.3 Advantages of ETD
• Preserva tion of PTMs: ETD preserves posttranslational modifications such as
phosphorylation, making it highly suitable for studying modified proteins and
peptides.
• Compatibility with high-mass analyzers: ETD works well with ion trap and other
mass analyzers used for high-mass biomolecules, making it versatile for largescale proteomics.
• Efficient fragmentation of large biomolecules: ETD is highly effective for
fragmenting large proteins, peptides, and other biomolecules that are difficult to
fragment using other methods.
9.3.2.6.2.4 Applications of ETD
• Proteomics: ETD is extensively used for sequencing peptides and proteins,
particularly for preserving labile PTMs such as phosphorylation and
glycosylation.
• Top-down proteomics: ETD is useful for analyzing intact proteins (top-down
proteomics) as it preserves the overall structure and modifications while
providing detailed fragmentation data.
• Study of biomolecule structure and folding: ETD provides insights into protein
folding and disulfide bonding by selectively fragmenting peptide backbones
while preserving side-chain information (Table
9.1).
9.3.2.7 Field Ionization (FI)
Field ionization (FI) is a soft ionization technique used in mass spectrometry to
generate ions from gaseous molecules without fragmenting them. This method is
primarily used for analyzing volatile and thermally stable compounds, particularly in
organic chemistry. It provides molecular ions with minimal fragmentation, making it
9.3 Instrumentation 375
Table 9.1 Comparison of ECD and ETD
Feature ECD ETD
Electron source Low-energy electrons Radical anions (electron transfer)
Fragment ions c-ions and z-ions c-ions and z-ions
Preservation of PTMs Yes Yes
Fragmentation efficiency Effective for high-charge-
Posttranslational
modifications (PTMs)
Primary application Proteomics, top-down
Instrumentation Often used with FT-ICR,
state ions
PTMs are preserved PTMs are preserved
analysis
Orbitrap
Effective for high-charge-state
ions
Proteomics, top-down, and
bottom-up analysis
Often used with ion traps and
Orbitrap
useful for determining the molecular weight of compounds while preserving the
structure of the analyte.
9.3.2.7.1 Principle of FI
• In field ionization, a high electric field is applied to a fine wire or needle with a
sharp tip (usually made of metal) placed in close proximity to the gaseous analyte.
• The intense electric field near the tip induces ionization of the gas-phase
molecules by pulling electrons from the molecules, creating positively
charged ions.
• The molecular ions are then directed into the mass analyzer for detection.
9.3.2.7.2 Key Features of FI
• Soft ionization: Field ionization is considered a “soft” ionization method, mean-
ing it produces ions with little to no fragmentation. This makes it ideal for
preserving the mol ecular structure of analytes.
• High voltage: The process requires an extremely high voltage (in the range of
6
–108 V/cm) to generate the electric field necessary for ionization.
10
• Minimal energy transfer: Since FI does not impart significant energy to the
molecule, fragmentation is minimal, making it suitable for analyzing intact
molecular ions.
9.3.2.7.3 Advantages of FI
• Molecular ion generation: FI produces molecular ions (M
+
) with minimal frag-
mentation, allowing for accurate molecular weight determination.
• Thermally stable compounds: It is especially useful for compounds that are
volatile and thermally stable, which may decompose in other ionization methods.
• Preserva tion of
structure: The gentle ionization method preserves the structure of
the analyte, which is important when structural integrity needs to be maintained
for identification.
376 9 Comprehensive Insights into Mass Spectrometry
9.3.2.7.4 Disadvantages of FI
• Limited use for nonvolatile compounds: FI is not suitable for nonvolatile, ther-
mally unstable, or large biomolecules.
• Requires high electric fields: The technique requires very high electric fields,
making it technically demanding and less versatile compared to other ionization
techniques.
9.3.2.7.5 Applications of FI
• Organic compound analysis: FI is used in organic chemistry to determine the
molecular weights of small, volatile organic compounds with minimal
fragmentation.
• Petrochemical analysis: FI is useful in analyzing hydrocarbons and other
petroleum-based compo unds, providing molecular ion data for complex mixtures.
• Structura l elucidation: FI helps in the identification of molecules by providing
molecular ion peaks that correspond to the intact analyte.
9.3.2.8 Desorption Electrospray Ionization (DESI)
Desorption electrospray ionization (DESI) is an ambient ionization technique used in
mass spectrometry that allows for the direct analysis of samples in their natural state
without the need for extensive sample preparation. It is widely used for the rapid
analysis of various materials, including biological tissues, surfaces, and forensic
evidence.
9.3.2.8.1 Principle of DESI
• In DESI, charged microdroplets of a solvent are sprayed onto a sample surface in
the open air.
• The solvent droplets impact the sample surface, desorbing and ionizing molecules
from the surface.
• The desorbed and ionized molecules are then carried into the mass spectrometer
for detection and analysis.
9.3.2.8.2 Key Features of DESI
• Ambient ionization: DESI operates under ambient
conditions,
meaning that
samples can be analyzed directly in their natural state, without requiring a vacuum
or complex sample preparation.
• Soft ionization: DESI is considered a soft ionization techni que, meaning it
generates ions with minimal fragmentation, preserving the molecular structure
of the analyte.
• Broad applicability: DESI is suitable for analyzing a wide variety of samples,
including solid, liquid, and semi-solid surfaces.
9.3.2.8.3 Advantages of DESI
•
Minimal sample
preparation: Samples can be analyzed directly, reducing the
time and effort required for sample preparation.
9.3 Instrumentation 377
• Nondestructive: Since DESI operates under ambient conditions, it often leaves
the sample relatively intact after analysis.
• Real-time analysis: DESI enables real-time analysis of surfaces and tissues,
making it useful for applications like in situ monitoring and rapid diagnostics.
• Wide range of applications: It is used in pharmaceutical analysis, forensics, food
safety, and even biomedical research, including tissue imaging.
9.3.2.8.4 Disadvantages of DESI
• Surface sensitivity: The effectiveness of DESI depends on the sample surface, and
rough or irregular surfaces may affect the efficiency of desorption and ionization.
• Lower sensitivity for complex matrices: While DESI can analyze complex
surfaces, its sensitivity can sometimes be limited in very intricate or heterogeneous matrices.
9.3.2.8.5 Applications of DESI
• Forensic science: DESI is used for the detection of explosives, drugs, and other
chemical residues on surfa ces such as clothing or paper, making it useful in
forensic investigations.
• Pharmaceutical industry: It allows for the rapid screening of pharmaceutical
compounds and
can analyze
drug formulations and active ingredients on tablets.
• Biological tissue imaging: DESI can create chemic al images of biological tissues,
allowing for the mapping of lipids, metabolites, and drugs in medical diagnostics
and research.
• Environmental monitoring: DESI is employed to analyze environmental samples
such as pollutants on surfaces and contaminants in food products.
9.3.2.9 Atmospheric Pressure Photoionization (APPI)
Atmospheric pressure photoionization (APPI) is an ionization technique used in
mass spectrometry, particularly for the analysis of nonpolar and moderately polar
compounds. APPI is an alternative to ESI and APCI, offering distinct advantages for
certain types of molecules.
9.3.2.9.1 Principle of APPI
• UV light source: In APPI, a vacuum ultraviolet (VUV) light source, usually a
krypton lamp, is used to ionize ana lyte molecules. The UV light photons ionize
the solvent or a dopant (a compound added to facilitate ionization), which then
transfers charge to the analyte molecules.
• Primary ionization: The
UV ligh
t produces radical cations from the solvent or
dopant molecules, which in turn ionize the analyte by either charge transfer or
proton transfer, depending on the ionization potential of the analyte.
• Ionization mechanism: APPI
+•
) and protonated molecules (MH+ ). This versatility allows it to analyze a
(M
mainly generates two types of ions: molecular ions
broader range of compounds, including those that are difficult to ionize with ESI
or APCI.
378 9 Comprehensive Insights into Mass Spectrometry
9.3.2.9.2 Key Features of APPI
• High efficiency for nonpolar compounds: APPI is particularly efficient for non-
polar and moderately polar compounds, such as hydrocarbons, steroids, and other
lipophilic molecules.
• Compatibility with LC-MS: APPI is commonly used
with liquid
chromatographymass spectrometry (LC-MS), making it a versatile tool for the analysis of
complex mixtures.
• Optional use of dopants: Dopants such as toluene, acetone, or anisole can be
introduced to enhance ionization efficiency by providing a more readily ionizable
substance that assists in charge transfer to the analyte.
9.3.2.9.3 Advantages of APPI
• Broad ionization range: APPI can ionize a wider range of chemical structures
than ESI and APCI, especially nonpolar compounds that are challenging to ionize
with other atmospheric pressure ionization techniques.
• Less matrix interference: APPI exhibits reduced matrix effects compared to ESI,
making it more suitable for samples with complex matrices such as biological
fluids or environmental samples.
• Combination with ESI/APCI: APPI can be easily combined with ESI and APCI in
the same mass spectrometer, offering flexibility for analyzing diverse sample
types.
9.3.2.9.4 Disadvantages of APPI
• Limited use for highly polar compounds: While APPI is excellent for nonpolar
and moderately polar analytes, it is less effective for highly polar compounds,
where ESI or APCI may be better suited.
• Requires dopants for certain analytes: For some compounds, dopants are neces-
sary to enhance ionization, adding complexity to the ionization process.
9.3.2.9.5 Applications of APPI
• Environmental analysis: APPI is used to detect and quantify nonpolar pollutants
such as polycyclic aromatic hydrocarbons (PAHs), pesticides, and persistent
organic pollutants (POPs) in environmental samples.
• Pharmaceutical analysis: In the pharmaceutical industry, APPI is employed for
analyzing lipophilic drug compounds, metabolites, and other challenging
substances that are not easily ionized by ESI.
• Petrochemical industry: APPI is useful for characterizing hydrocarbons, oils, and
other nonpolar compounds in the petrochemical industry.
• Lipidomics: APPI is also applied in lipidomics research,
where it
aids in the
analysis of nonpolar lipids that are difficult to ionize using traditional methods.
9.3.2.9.6 Comparison of ESI, APCI, and APPI
Features ESI APCI APPI
Ionization
mechanism
Produces ions by
applying a high voltage
a liquid sample,
to
Involves ionization
through chemical
reactions
between the
Utilizes UV light to
ionize analytes in the
gas phase, generating
(continued)
9.3 Instrumentation 379
Features ESI APCI APPI
creating a fine aerosol
that is then ionized as it
passes through a heated
desolvation chamber
Sample state Suitable for liquid
samples (solutions and
suspensions)
Molecular
weight range
Effective for a wide
range of molecular
weights, typically from
small molecules to large
biomolecules (up to
200 kDa)
Ion types Primarily produces
protonated (M + H)
and deprotonated (M -
-
ions; can also
H)
generate adduct ions
analyte and ions
produced by a corona
discharge in the
presence of a solvent
Suitable for liquid
samples, particularly
those with
polarities
Best for small- to
medium-sized
molecules (up to a few
kDa), often less
effective for very large
biomolecules
Produces mostly
+
protonated or
deprotonated ions,
along with ion clusters;
ionization efficiency
may vary based on
lower
ions through the
photoionization process
Suitable for
both polar
and nonpolar liquid
samples and some
solids
Typically, effective for
small- to medium-sized
molecules, but also
accommodates larger
analytes
Produces both
protonated and neutral
ions; often generates
different ion types
depending on the UV
wavelength used
solvent composition
Sensitivity Highly sensitive for
polar and nonpolar
compounds, particularly
suitable for
biomolecules like
Moderate sensitivity,
often less sensitive than
ESI, but can be
enhanced through
method optimization
Generally, provides
high sensitivity,
especially for nonpolar
and aromatic
compounds
proteins and peptides
Speed Provides rapid analysis;
suitable for highthroughput applications
Typically, slower than
ESI, especially for
volatile analytes;
however, it is relatively
Offers quick analysis
times; comparable to
ESI in many
applications
fast for moderate
polarity compounds
Temperature
sensitivity
Applications Widely used in
Limitations Limited effectiveness
Sensitive to
temperature, requiring
controlled conditions to
maintain analyte
stability
proteomics,
metabolomics,
and
pharmaceutical
analysis; excellent
for
studying large
biomolecules
nonpolar
for
and
volatile compounds;
potential issues with ion
suppression in complex
Generally, less sensitive
to temperature
variations; heat
applied
is often
to enhance
desolvation
Commonly used for
environmental analysis,
pharmaceuticals, and
small organic
compounds; often
applied in drug
metabolism studies
ot b
May n
e effective for
polar compounds;
requires careful solvent
selection for optimal
ionization
Temperature-sensitive;
optimal ionization
requires controlled
temperature and light
conditions
Applied in
environmental analysis,
pharmaceutical testing,
and the analysis of
complex matrices;
effective for analyzing
nonpolar compounds
Less effective
for highly
polar or very large
biomolecules; UV light
can cause fragmentation
of sensitive analytes
mixtures
380 9 Comprehensive Insights into Mass Spectrometry
9.3.2.10 Matrix-Assisted Laser Desorption/Ionization (MALDI)
Matrix-assisted laser desorption/ionization (MALDI) is a powerful ionization technique used in mass spectrometry, particularly suitable for the analysis of large
biomolecules such as proteins, peptides, nucleic acids, and polysaccharides.
MALDI allows for the generation of intact ions from these fragile molecules without
fragmentation, making it ideal for high-mass analytes.
9.3.2.10.1 Principle of MALDI
• Matrix selection: The technique involves mixing the analyte of interest with a
suitable organic matrix, which absorbs the energy from a laser pulse. Common
matrices include α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid, and
2,5-dihydroxybenzoic acid (DHB).
• Sample preparation: The analyte–matrix mixture is then applied to a target plate
and allowed to dry, forming a crystalline layer. During the desorption/ionization
process, the matrix crystals assist in the energy transfer from the laser to the
analyte.
• Laser irradiation: A laser (commonly a nitrogen laser emitting at 337 nm or a
solid-state laser) is focused onto the sample. The laser energy causes the matrix to
rapidly vaporize, leading to the desorption of the analyte molecules into the gas
phase.
• Ion formation: The matrix can also ionize the analyte, typically by forming
protonated or deprotonated ions (M + H
+
or M - H-). This ionization occurs
in the gas phase, allowing for the generation of intact molecular ions.
9.3.2.10.2 Key Features of MALDI
• Soft ionization: MALDI is considered a soft ionization technique because it
minimizes fragmentation of the analyte, allowing for the preservation of the intact
molecular weight.
• High mass range: MALDI can effectively analyze large biomolecules, with mass
ranges extending to several hundred kilodaltons (kDa) or even megadaltons
(MDa).
• Rapid analysis: The technique allows for rapid sample analysis, making it
suitable for high-throughput applications.
9.3.2.10.3 Advantages of MALDI
• Minimal sample preparation: The sample preparation process is straightforward
and requires minimal steps, making it accessible for a wide range of applications.
• Versatility: MA
LDI can analyz
e various sample types, including proteins,
peptides, nucleic acids, and synthetic polymers, making it widely applicable in
biological and biomedical research.
• Detection of postt
ranslational modifications: The technique is useful for
identifying and characterizing posttranslational modifications (PTMs) of proteins,
such as phosphorylation, glycosylation, and acetylation.