QPNC-PAGE

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QPNC-PAGE, or QuantitativePreparativeNativeContinuousPolyacrylamideGel Electrophoresis, is a bioanalytical, one-dimensional, high-resolution and high-precision electrophoresis technique applied in biochemistry and bioinorganic chemistry to quantitatively separate proteins by isoelectric point and by continuous elution from a gel column. [1]

Contents

This hybrid variant of native analytical gel electrophoresis and preparative polyacrylamide gel electrophoresis is used by biologists to resolve low concentrations of macromolecules with high recovery, for example, into active or native metalloproteins in biological samples or into properly and improperly folded metal cofactor-containing proteins in complex protein mixtures. [2]

Introduction

Proteins perform several functions in living organisms, including catalytic reactions and transport of molecules or ions within the cells, the organs or the whole body. The understanding of the processes in human organisms, which are mainly driven by biochemical reactions and protein-protein interactions, depends to a great extent on the ability to isolate active proteins in biological samples for more detailed examination of chemical structure and physiological function. This essential information can imply an important indication of a patient's state of health. [3]

As about 30–40% of all known proteins contain one or more metal ion cofactors (e.g., ceruloplasmin, ferritin, amyloid-beta precursor protein, matrix metalloproteinase, or metallochaperones), especially native and denatured metalloproteins have to be isolated, identified and quantified after liquid biopsy. Many of these cofactors (e.g., iron, copper, or zinc) play a key role in vital enzymatic catalytic processes or stabilize globular protein molecules. [4] Therefore, the high-precision gel electrophoresis and comparable separation techniques are highly relevant as initial step of protein and trace metal speciation analysis, subsequently, followed by modern mass spectrometric and magnetic resonance methods for quantifying and identifying the soluble proteins of interest. [5]

Method

Separation and buffering mechanisms

Equipment for bioanalytical continuous-elution gel electrophoresis: electrophoresis chamber, peristaltic pump, fraction collector, buffer recirculation pump and UV detector (in a refrigerator), power supply and recorder (on a table). Electrphoresis Tools.TIF
Equipment for bioanalytical continuous-elution gel electrophoresis: electrophoresis chamber, peristaltic pump, fraction collector, buffer recirculation pump and UV detector (in a refrigerator), power supply and recorder (on a table).

In gel electrophoresis proteins are normally separated by charge, size, or shape. [7] The aim of isoelectric focusing (IEF), for example, is to separate proteins according to their isoelectric point (pI), thus, according to their charge at different pH values. [8] Here, a similar mechanism is accomplished in a commercially available electrophoresis chamber for separating charged biomolecules, for example, superoxide dismutase (SOD) [9] or allergens, [10] at constant pH conditions and different velocities of migration depending on different isoelectric points of zwitterions. The separated (metal) proteins elute sequentially, starting with the lowest (pI > 2–4) and ending with the highest pI (pI < 10.0) of the dissolved protein molecules to be analyzed. [11]

Due to the specific properties of the prepared gel and electrophoresis buffer solution which is basic and contains Tris-HCl and NaN3, [6] most proteins of a biological system (e.g., Helicobacter pylori [12] ) are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field. In general, reaction equation (1) shows that the carboxyl side group of a proteinogenic amino acid is negatively charged, equation (2) that the amino side groups are electrically neutral under these conditions:

(1) R-COOH + OH− → R-COO− + H2O

(2) R-NH3+ + OH− → R-NH2 + H2O

At the anode, electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions (3). The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water (4):

(3) (HOCH2)3CNH2 + H+ → [(HOCH2)3CNH3]+

(4) [(HOCH2)3CNH3]+ + OH− → (HOCH2)3CNH2 + H2O

Thus, the Tris-based buffering mechanism causes a constant pH in the continuous buffer system with a high buffer capacity. [13]

At 25 °C Tris buffer has an effective pH range between 7.5 and 9.0. Under the conditions given here (addressing the concentration of buffer components, buffering mechanism, pH and temperature) the effective pH is shifted in the range of about 10.0 to 10.5. Native buffer systems all have low conductivity and range in pH from 3.8 to 10.2. Continuous native buffer systems are thus used to separate proteins according their pI. [14]

Although the pH value (10.00) of the electrophoresis buffer does not correspond to a physiological pH value within a cell or tissue type, the separated ring-shaped protein bands are eluted continuously into a physiological buffer solution (pH 8.00) and isolated in different fractions. [6] Provided that irreversible denaturation cannot be demonstrated by an independent procedure, most protein molecules are stable in aqueous solution, at pH values from 3 to 10 if the temperature is below 50 °C. [15] As the Joule heat and temperature generated during electrophoresis may exceed 50 °C, [16] and thus, have a negative impact on the stability and migration behavior of proteins in the gel, the separation system, consisting of the electrophoresis chamber, fraction collector and other devices, is cooled in a refrigerator at 4 °C, thus, greatly reducing the risk of heat convection currents. [17] Overheating of the gel is impeded by internal cooling circuit of the gel column as an integrated part of the electrophoresis chamber and by generating a constant power by the power supply. [18]

Gel properties and polymerization time

Best polymerization conditions for acrylamide gels are obtained at 25–30 °C [19] and polymerization seems terminated after 20–30 min of reaction although residual monomers (10–30%) are detected after this time. [20] The co-polymerization of acrylamide (AA) monomer/N,N'-Methylenebisacrylamide (Bis-AA) cross-linker initiated by ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) reactions, is most efficient at alkaline pH of the acrylamide solution. Thereby, acrylamide chains are created and cross-linked at a time. Due to the properties of the electrophoresis buffer, the gel polymerization is conducted at pH 10.00 making sure an efficient use of TEMED and APS as catalysts of the polymerization reaction, and concurrently, suppressing a competitive hydrolysis of the produced acrylamide polymer network. Polymer networks are three-dimensionally linked polymer chains. Otherwise, proteins could be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products that may result in multiple bands. [21]

Additionally, the time of polymerization of a gel can directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression and dilatation of the gels and their pores if the incubation times for the reaction mixture (gel solution) used to prepare a gel are not optimized. In order to ensure maximum reproducibility in gel pore size and to obtain a fully polymerized and non-restrictive large pore gel for a PAGE run, the polyacrylamide gel is polymerized for a time period of 69 hr at room temperature (RT) in a gel column located on the casting stand. [18] The exothermic heat generated by the polymerization processes is dissipated constantly while the temperature may rise rapidly to over 75 °C in the first minutes, after which it falls slowly. [22] After 69 hr, the gel has reached room temperature and is in its lowest energy state, as the basic chemical reactions and gelation are complete. [18] Gelation means that the solvent (water) gets immobilized within the polymer network by means of hydrogen bonds and also van der Waals forces. As a result, the prepared gel is homogeneous (in terms of homogeneous distribution of cross-links throughout the gel sample [23] ), inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins. [24] Sieving effects due to gravity-induced compression of the gel can be excluded for the same reasons. Thus, in a medium without molecular sieving properties a high-resolution can be expected. [25]

Before an electrophoretic run is started the prepared 4% T (total polymer content (T)), 2.67% C (cross-linker concentration (C)) gel is pre-run to equilibrate it. [6] It is essentially non-sieving and optimal for electrophoresis of proteins greater than or equal to 200 ku. Proteins migrate in it more or less on the basis of their free mobility. [26] For these reasons interactions of the gel with the biomolecules are negligibly low, and thus, the proteins separate cleanly and predictably at a polymerization time of 69 hr. The separated metalloproteins including biomolecules ranging from approximately < 1 ku to greater than 30 ku (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides, metallothionein, phytochelatins) are not dissociated into apoproteins and metal cofactors. [27]

Reproducibility and recovery

Electropherogram showing four PAGE runs of a high molecular weight plant protein ([?] 200 kDa) as a function of time of polymerization of the gel. Detection method for determination of Cd cofactor concentrations (in mg/L): GF-AAS. Preparative Electroph.TIF
Electropherogram showing four PAGE runs of a high molecular weight plant protein (≈ 200 kDa) as a function of time of polymerization of the gel. Detection method for determination of Cd cofactor concentrations (in μg/L): GF-AAS.

The bioactive structures (native or 3D conformation or shape) of the isolated protein molecules do not undergo any significant conformational changes. Thus, active metal cofactor-containing proteins can be isolated reproducibly in the same fractions after a PAGE run. [11] A shifting peak in the respective electropherogram indicates that the standardized time of gel polymerization (69 hr, RT) is not implemented in a PAGE experiment. A lower deviation of the standardized polymerization time (< 69 hr) stands for incomplete polymerization, and thus, for inherent instability due to gel softening during the cross-linking of polymers as the material reaches swelling equilibrium, [28] whereas exceeding this time limit (> 69 hr) is an indicator of gel aging. [29] The phenomenon of gel aging is closely connected to long-term viscosity decrease of aqueous polyacrylamide solutions [30] and increased swelling of hydrogels. [31]

Under standard conditions, metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a quantitative yield of more than 95%. [18] By preparative SDS-PAGE standard proteins (cytochrome c, aldolase, ovalbumin and bovine serum albumin) with molecular masses of 14–66 ku can be recovered with an average yield of about 73.6%. [32] Preparative isotachophoresis (ITP) is applied for isolating palladium-containing proteins with molecular masses of 362 ku (recovery: 67%) and 158 ku (recovery: 97%). [33]

Quantification and identification

Physiological concentrations (ppb-range) of Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn, Pt, Cr, Cd and other metal cofactors can be identified and absolutely quantified in an aliquot of a fraction by inductively coupled plasma mass spectrometry (ICP-MS) [34] or total reflection X-ray fluorescence (TXRF), [35] for example. In case of ICP-MS the structural information of the associated metallobiomolecules is irreversibly lost due to ionization of the sample with plasma. [36] [37] Another established high sensitive detection method for the determination of trace elements in biological samples is graphite furnace atomic absorption spectrometry (GF-AAS). [38] Because of high purity and optimized concentration of the separated metalloproteins, for example, therapeutic recombinant plant-made pharmaceuticals such as copper chaperone for superoxide dismutase (CCS) from medicinal plants, in a few specific PAGE fractions, the related structures of these bioactive analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions. [39]

Applications

Improperly folded metal proteins, for example, CCS or Cu-Zn-superoxide dismutase (SOD1) present in brain, blood or other clinical samples, are indicative of neurodegenerative diseases like Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS). [40] Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ion species (e.g., Cu1+/2+, Zn2+, Fe2+/3+, Mn2+, Ni3+) in organisms, and thus, these biomolecules can balance pro-oxidative and antioxidative processes in the cytoplasm. [41] Otherwise, free or loosely bound transition metal ions take part in Fenton-like reactions in which deleterious hydroxyl radical is formed, which unrestrained would be destructive to proteins. [42] The loss of active CCS increases the amyloid-β production in neurons which, in turn, is a major pathological hallmark of AD. [43] Therefore, copper chaperone for superoxide dismutase is proposed to be one of the most promising biomarkers of Cu toxicity in these diseases. [44] CCS should be analysed primarily in blood because a meta-analysis of serum data showed that AD patients have higher levels of serum Cu than healthy controls. [45]

Persons

QPNC-PAGE, originally ‘PNC-PAGE’, [46] was invented and developed in Germany in the late 1990s and was significantly influenced by the pioneering work of David E. Garfin. [47]

See also

Related Research Articles

<span class="mw-page-title-main">Gel electrophoresis</span> Method for separation and analysis of biomolecules

Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also used. For brevity, this article uses pI. The net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).

<span class="mw-page-title-main">Superoxide dismutase</span> Class of enzymes

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O−
2) anion radical into normal molecular oxygen (O2) and hydrogen peroxide (H
2O
2). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, causes many types of cell damage. Hydrogen peroxide is also damaging and is degraded by other enzymes such as catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use intracellular manganese to prevent damage from reactive O−
2.

<span class="mw-page-title-main">Polyacrylamide gel electrophoresis</span> Analytical technique

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

<span class="mw-page-title-main">Metalloprotein</span> Protein that contains a metal ion cofactor

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins contain zinc-binding protein domains although there may be up to 3000 human zinc metalloproteins.

<span class="mw-page-title-main">Electrophoresis</span> Motion of charged particles in electric field

Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions.

<span class="mw-page-title-main">Polyacrylamide</span> Chemical compound

Polyacrylamide (abbreviated as PAM or pAAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly for water treatment and the paper and mineral industries.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Gel electrophoresis of proteins</span> Technique for separating proteins

Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The electrophoresis may be performed with a small volume of sample in a number of alternative ways with or without a supporting medium, namely agarose or polyacrylamide. Variants of gel electrophoresis include SDS-PAGE, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each variant has many subtypes with individual advantages and limitations. Gel electrophoresis is often performed in combination with electroblotting or immunoblotting to give additional information about a specific protein.

<span class="mw-page-title-main">Coomassie brilliant blue</span> Chemical compound

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<span class="mw-page-title-main">Isoelectric focusing</span> Type of electrophoresis

Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other electrophoretic techniques including capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC) belong also to this class of methods. In CE methods, analytes migrate through electrolyte solutions under the influence of an electric field. Analytes can be separated according to ionic mobility and/or partitioning into an alternate phase via non-covalent interactions. Additionally, analytes may be concentrated or "focused" by means of gradients in conductivity and pH.

<span class="mw-page-title-main">Electroelution</span>

Electroelution is a method used to extract a nucleic acid or a protein sample from an electrophoresis gel by applying a negative current in the plane of the smallest dimension of the gel, drawing the macromolecule to the surface for extraction and subsequent analysis. For example, electroblotting and preparative native PAGE are based upon the same principle.

<span class="mw-page-title-main">MOPS</span> Chemical compound

MOPS (3-(N-morpholino)propanesulfonic acid) is a buffer introduced in the 1960s, one of the twenty Good's buffers. It is a structural analog to MES, and like MES, its structure contains a morpholine ring. HEPES is a similar pH buffering compound that contains a piperazine ring. With a pKa of 7.20, MOPS is an excellent buffer for many biological systems at near-neutral pH.

Within chemistry for acid–base reactions, Immobilized pH gradient (IPG) gels are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. The immobilized pH gradient is obtained by the continuous change in the ratio of Immobilines. An Immobiline is a weak acid or base defined by its pK value. Immobilized pH gradients (IPG) are made by mixing two kinds of acrylamide mixture, one with Immobiline having acidic buffering property and other with basic buffering property. The concentrations of the buffers in the two solutions define the range and shape of the pH gradient produced. Both solutions contain acrylamide monomers and catalysts. During polymerization, the acrylamide portion of the buffers co polymerize with the acrylamide and bisacrylamide monomers to form a polyacrylamide gel. These polymerised gels are backed with plastic based backing that allow ease in handling and improve IPG's performance. The gel is then washed to remove catalysts and unpolymerized monomers, which interfere with isoelectric separation. IPG increased reproducibility of isoelectric focusing and 2D-gel electrophoresis. Other advantages are increased resolution, reproducible separation of alkaline proteins and increased loading capacity.

<span class="mw-page-title-main">Affinity electrophoresis</span>

Affinity electrophoresis is a general name for many analytical methods used in biochemistry and biotechnology. Both qualitative and quantitative information may be obtained through affinity electrophoresis. Cross electrophoresis, the first affinity electrophoresis method, was created by Nakamura et al. Enzyme-substrate complexes have been detected using cross electrophoresis. The methods include the so-called electrophoretic mobility shift assay, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the electrophoretic pattern of molecules through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent. Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules. The methods have been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one-dimensional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Some of the methods are similar to affinity chromatography by use of immobilized ligands.

Free-flow electrophoresis (FFE), also known as carrier-free electrophoresis, is a matrix-free, high-voltage electrophoretic separation technique. FFE is an analogous technique to capillary electrophoresis, with a comparable resolution, that can be used for scientific questions, where semi-preparative and preparative amounts of samples are needed. It is used to quantitatively separate samples according to differences in charge or isoelectric point by forming a pH gradient. Because of the versatility of the technique, a wide range of protocols for the separation of samples like rare metal ions, protein isoforms, multiprotein complexes, peptides, organelles, cells, DNA origami, blood serum and nanoparticles exist. The advantage of FFE is the fast and gentle separation of samples dissolved in a liquid solvent without any need of a matrix, like polyacrylamide in gel electrophoresis. This ensures a very high recovery rate since analytes do not adhere to any carrier or matrix structure. Because of its continuous nature and high volume throughput, this technique allows a fast separation of preparative amounts of samples with a very high resolution. Furthermore, the separations can be conducted under native or denaturing conditions.

<span class="mw-page-title-main">Discontinuous electrophoresis</span> Type of laboratory technique

Discontinuous electrophoresis is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis. This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.

<span class="mw-page-title-main">SDS-PAGE</span> Biochemical technique

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. The combined use of sodium dodecyl sulfate and polyacrylamide gel eliminates the influence of structure and charge, and proteins are separated by differences in their size. At least up to 2012, the publication describing it was the most frequently cited paper by a single author, and the second most cited overall.

<i>N</i>,<i>N</i>-Diallyl-L-tartardiamide Chemical compound, polyacrylamide crosslinker

N,N′-Diallyl-L-tartardiamide (DATD) is a crosslinking agent for polyacrylamide gels, e.g., as used for SDS-PAGE. Compared to bisacrylamide gels, DATD gels have a stronger interaction with glass, and therefore are used in applications where the polyacrylamide gel acts as a "plug" structural component at the bottom of a gel electrophoresis apparatus, thereby preventing a weak discontinuous gel from sliding out from or otherwise moving within the apparatus. Unlike bisacrylamide-polyacrylamide gels, DATD-polyacrylamide gels can be conveniently dissolved using periodic acid due to the presence of viscinal diols in DATD. DATD is the slowest polyacrylamide crosslinker tested, and has can act as an inhibitor of polymerization at high concentrations.

References

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