Другое на тему СAPILIARY ELECTROFORESIS COUPLED TO MASS SPECTROMETRY FOR STUDING INTACT PROTEINS (Капилиарный электрофорез в связи с масс-спектрометрией для исследования интактных белков)

Содержание:

1. Introduction. 3

2. CE of proteins. 5

2.1. CE modes. 5

2.2. Capillary coatings. 8

2.3. Pretreatment and preconcentration techniques. 14

3. CE–MS coupling for proteins. 18

3.1. Ionization techniques and interfacing. 18

3.1.1. Electrospray ionization (ESI) 18

3.1.2. Matrix assisted laser-desorption ionization
(MALDI) 19

3.1.3. Inductively coupled plasma ionization (ICP) 20

3.2. MSs. 21

4. Applications of CE–MS for intact protein
analysis. 24

4.1. Capillary zone electrophoresis–mass
spectrometry (CZE-MS) 24

4.1.1. Electrospray ionization (ESI) 24

4.1.2. Matrix assisted laser-desorption ionization
(MALDI) 28

4.2. 2 D-gel Electrophoresis-Mass Spectrometry
(2D-gel Electrophoresis-MS) 30

4.2.1. Inductively coupled plasma ionization (ICP) 30

4.3. Chip-based electrophoresis coupled with mass
spectrometry (Chip/CE-MS) 34

4.3.1. Electrospray ionization (ESI) 34

Conclusion. 40

References  42  

Введение:

Developments
in the fields of protein chemistry, proteomics and biotechnology have caused an
increasing demand for sensitive and selective analytical tools for the analysis
of intact proteins. Today, various separation techniques are available for the
qualitative and quantitative analysis of proteins. Frequently used techniques
are slab-gel electrophoresis (SGE), liquid chromatography (LC) and capillary
electrophoresis (CE) [1]. SGE is an established technique for protein
separation, as many samples can be analyzed simultaneously and a relatively
large number of proteins can be resolved applying two-dimensional SGE. However,
limitations are the relatively long and labor-intensive analysis, the necessity
of off-line detection, and the lack of precise (and automated) quantitation. LC
is advantageous due to its separation power, ease of automation and routine
coupling with various detection principles, like mass spectrometry (MS).

On the
other hand, adverse interactions of proteins with the stationary phase and
denaturation of the proteins in organic mobile phases can seriously hinder LC
analyses and decrease separation efficiency and resolution. CE offers
attractive features for the analysis of proteins, as the analysis times can be
relatively short and only minute amounts of sample are needed. Furthermore,CE analyses
are carried out in fused-silica capillaries under aqueous conditions and in the
absence of a stationary phase. This enables the study of proteins without
causing conformational changes due to organic modifiers and/or a stationary
phase. In addition, separation conditions inCEcan be chosen in such away that
separations can be performed under (near-)physiological conditions avoiding
protein degradation during analysis. Finally, as the CE separation is a
function of charge, size and shape of a compound, small differences in the size
or charge of proteins may be sufficient for separation, especially with the
high efficiencies normally obtained in CE.

The object
of the study is СЕ of intact proteins.

The subject
of the research is СЕ in connection with mass spectrometry for the study of
intact proteins.

In this
study different articles base d on CE in combination with MS used for intact
proteins will be discussed base don the following points:

— CE modes.

— Capillary
coatings.

—
Pretreatment and preconcentration techniques.

—
Ionization techniques and interfacing.

— MSs.

— Capillary
zone electrophoresis–mass spectrometry (CZE-MS).

— 2 D-gel
Electrophoresis-Mass Spectrometry (2D-gel Electrophoresis-MS).

—
Chip-based electrophoresis coupled with mass spectrometry (Chip/CE-MS).

The
structure of the work is presented by an introduction, three sections, a
conclusion and a list of references.

Заключение:

A real
breakthrough in mass spectrometry of peptides was associated with the creation
of the fast atom bombardment method in the mid-1970s. It became possible to
establish the molecular masses and the sequence of amino acid units of
oligopeptides with masses of one thousand or more Daltons. 15 years later, with
the advent of electrospray ionization and matrix-assisted laser desorption /
ionization, mass spectrometry became the key method for determining the primary
structure of peptides. A new science appears — proteomics.

Mass
spectrometry is one of the most useful CEC detection methods because it offers
many advantages over other traditional detection methods. Most importantly, MS
provides information on the chemical structure and molecular weight of the
analytes. Electrospray Ionization (ESI), which generates gas phase ions from
analytes dissolved in a liquid phase, is a soft interface ionization technique
for linking CEC separations to MS. Thus, CEC-ESI-MS is a promising method for
the simultaneous separation and detection of biomolecules because it combines
the advantages of high separation efficiency and chromatographic selectivity in
CEC and high sensitivity and chemical and molecular mass information in MS
detection.

All
proposed methods of absolute quantitative determination are effective in the
case of reliable detection of labeled and unlabeled peptides in the analyzed
mixture. At the same time, the sample, as a rule, contains many other proteins,
or few other proteins, but their concentration significantly exceeds the
concentration of the analyzed protein. In this case, there is a high risk of
"missing" the target peptide during standard or rapid-fire CEC-ESI-MS
analysis. In this regard, when working with the internal standard. In this
case, the MS selectively records the peaks of only labeled / unlabeled peptides
(or their set in the case of PSAQ).

In the case
of MALDI, the quantification of proteins is even more difficult, since the
absolute intensities of the characteristic ion peaks depend on the chemical
nature of the analytes, the presence of other proteins and impurities in the
sample, as well as numerous analysis parameters. Therefore, it becomes
imperative to use a reliable internal standard.

Фрагмент текста работы:

2.
CE of proteins

2.1.
CE modes

СЕ, CE is a
separation method implemented in capillaries and based on the differences in
electrophoretic mobility of charged particles in both aqueous and non-aqueous
buffer electrolytes. Buffer solutions (leading electrolytes, working buffers,
background electrolyte, run buffer) may contain additives for example,
macrocycles, organic solvents, polymers,  that can interact with the analyzed particles
and change their electrophoretic mobility.

The use of
the term СЕas a general term for all methods of СЕis not recommended, since
many of these methods (capillary gel electrophoresis, capillary affinity
electrophoresis, capillary isoelectric focusing, capillary isotachophoresis,
micellar electrokinetic chromatography, emioncinematic chromatography) separation
without non-CE separation principles.

Traditionally,
СЕis compared to high performance liquid chromatography (HPLC), since in both
methods the separation occurs in a confined space (capillary or column) with
the participation of a moving liquid phase (buffer solution or mobile phase
(eluent)) and similar detection principles are used to record signals and data
processing programs. Nevertheless, the methods have differences, which
undoubtedly relate to the advantages of СЕ[26]:

— high
separation efficiency (hundreds of thousands of theoretical plates),
inaccessible to HPLC and associated with the flat EOF profile,

— small
volume of the analyzed sample and buffers (no more than 1–2 ml per day), while
the use of high-purity, expensive organic solvents is practically not required,

— absence
of a column, sorbent, problems with aging and, therefore, replacement of the
column,

— simple
and inexpensive equipment,

— rapidity
and low cost of a single analysis.

Among the
CE restrictions, it should be noted that, in comparison with High-performance
liquid chromatography (HPLC), the concentration sensitivity is low and the
requirement for the analyzed compounds to dissolve in water and dilute
aqueous-organic mixtures. At the same time, these restrictions are not
insurmountable. Thus, the insufficient sensitivity of the determination when
using UV detection (due to the small length of the optical path equal to the
inner diameter of the capillary) can be compensated for by the use of such
types of detection as laser-induced fluorimetric or mass spectrometric in
combination with various methods of on-line concentration of the sample. (the
so-called stacking and sweeping). And the variant of non-aqueous СЕsuccessfully
allows the separation and analysis of highly hydrophobic, insoluble in aqueous
solutions, sample components.

The method
of СЕis today successfully used for the analysis of various substances, inorganic
and organic cations and anions, amino acids, vitamins, drugs, dyes, proteins,
etc. Оbjects, for monitoring the quality of water and drinks, technological
control of production, incoming control raw materials, analysis of
pharmaceuticals, and food products, in
forensics, medicine, biochemistry, etc. [9].

In the
method of СЕ, open systems are used as follows. The electrolyte solution in
which the separation takes place is not separated from the electrodes to which
a voltage is applied. The near-electrode spaces are connected through a thin
quartz capillary that performs the main separating function, but serves as an
electrolytic bridge. Сlosing
the electrical circuit. In electrical circuits containing simultaneously
conductors of the first and second kind, current flow is impossible without
electrochemical reactions at the metal-solution boundaries. In СЕ, they try to
use such compositions of buffer leading electrolytes, in which water decomposes
on the electrodes (one of the most common buffers for CE is borax solution). At
the cathode, hydrogen ions are reduced, molecular hydrogen is released on the
cathode surface and hydroxyl ions are formed in the cathode space. At the anode
— the oxidation of hydroxyl ions, the release of molecular oxygen on the anode
surface and the formation of hydrogen ions in the anode space [17].

At the
cathode:

At the
anode:

At high
potential differences, which are used in CE, other parallel electrochemical
reactions can occur on the electrodes, but the above are the main ones.

The
simplest CE option is Capillary Zone Electrophoresis, CZE.

The
components of a complex mixture move in the electrolyte medium at different
speeds, forming discrete zones. A distinctive feature of CZE is that it is
suitable for separating only ionic components of a sample, while neutral compounds
that do not have their own electrophoretic mobility move at an EOF speed and
exit in the zone of neutral components, the zone of the EOF marker [6].

Micellar
electrokinetic chromatography combines electrophoresis and chromatography.
Introduced in 1984 by the Japanese scientist Terabe, MEKC has become the most
widespread among other variants of СЕ, primarily due to the ability to separate
both ionic and uncharged sample components. The separation of neutral compounds
became possible due to the introduction of anionic surfactants (surfactants) —
micelle-forming agents into the leading electrolyte. Most often, anionic
surfactants (for example, sodium dodecyl sulfate — SDS) are used in
concentrations exceeding the critical micelle concentration (CMC), which, for
example, for SDS in an aqueous solution is 8 mM. In this case, the electrolyte
solution contains mainly micelles and a small fraction of the monomeric form of
the surfactant. Monomers consist of a hydrophobic "tail" and a
hydrophilic (in the case of an anionic surfactant, negatively charged)
"head". During the formation of straight micelles, monomeric
fragments aggregate with non-polar ends inward, and the outer spherical surface
of the micelle becomes negatively charged. Each micelle is surrounded by its
own double electric layer, the outer diffuse part of which is formed by cations
present in the leading electrolyte solution. The number of monomers forming a
micelle can vary from 60 to 100 molecules; however, the total charge of a
micelle is significantly lower due to the presence of hydrated