QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a high-resolution and a highly accurate technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively by isoelectric point. This standardized variant of native gel electrophoresis is used by biologists to isolate active or native metalloproteins in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins or protein isoforms in complex protein mixtures. A high reproducibility in gel pore size and a high-yield electroelution of proteins performed by this highly reliable technique strongly correlate with the polymerization time of the acrylamide gels representing an inherent stability constant of the separation system.
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 organisms, which are mainly driven by biochemical reactions and protein-protein interactions, depends to a great extent on our ability to isolate active proteins in biological samples for more detailed examination of chemical structure and physiological function. Because this information can be an important indication of a patient's state of health.
As about 30-40% of all known proteins contain one or more metal ion cofactors (e.g., ceruloplasmin, ferritin, amyloid precursor protein, matrix metalloproteinase), especially native 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. Therefore, the high-precision electrophoresis and other native separation techniques are highly relevant as initial step of protein analysis and trace metal speciation followed by mass spectrometric and magnetic resonance methods for quantifying and identifying the soluble proteins of interest.
Separation and buffering mechanisms
In gel electrophoresis proteins are normally separated by charge, size, or shape. 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. Here, the same mechanism is accomplished in a commercially available electrophoresis chamber (see figure Equipment) for separating charged biomolecules, for example, superoxide dismutase (SOD) or allergens, at continuous pH conditions and different velocities of migration depending on different isoelectric points. The separated (metal) proteins elute sequentially, starting with the lowest (pI > 2-4) and ending with the highest pI (pI < 10.0) of the present protein molecules.
Due to the specific properties of the prepared gel and electrophoresis buffer solution (which is basic and contains Tris-HCl and NaN3), most proteins of a biological system (e.g., Helicobacter pylori) are charged negatively in the solution, and will migrate from the cathode to the anode due to the electric field. At the anode, electrochemically-generated hydrogen ions react with Tris molecules to form monovalent Tris ions. The positively charged Tris ions migrate through the gel to the cathode where they neutralise hydroxide ions to form Tris molecules and water. Thus, the Tris-based buffering mechanism causes a constant pH in the buffer system. At 25 °C Tris buffer has an effective pH range between 7.5 and 9.0. Under the conditions given here (addressing the concentration, 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.
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 (see figure Electropherogram). 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. As the Joule heat and temperature generated during electrophoresis may exceed 50 °C, and thus, have a negative impact on the stability and migration behavior of proteins, the separation system, including the electrophoresis chamber and a fraction collector, is cooled in a refrigerator at 4 °C. Overheating of the gel is impeded by internal cooling of the gel column and by generating a constant power (see figure Equipment).
Gel properties and polymerization time
Best polymerization conditions for acrylamide gels are obtained at 25-30 °C and polymerization seems terminated after 20-30 min of reaction although residual monomers (10-30%) are detected after this time. The co-polymerization of acrylamide (AA)/N,N'-Methylenebisacrylamide (Bis-AA) initiated by ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) reactions, is most efficient at alkaline pH. 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 (cf. section Principle). Otherwise, proteins could be modified by reaction with unpolymerized monomers of acrylamide, forming covalent acrylamide adduction products that may result in multiple bands.
Additionally, the time of polymerization of a gel may directly affect the peak-elution times of separated metalloproteins in the electropherogram due to the compression and dilatation of the gels and their pores with the longer incubation times (see figure Electropherogram, cf. subsection Reproducibility and recovery). 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). 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. After 69 hr the gel has reached room temperature, and thus, is in its lowest energy state because the chemical reactions and the gelation are terminated. As a result, the prepared gel is homogeneous (in terms of homogeneous distribution of cross-links throughout the gel sample), inherently stable and free of monomers or radicals. Fresh polyacrylamide gels are further hydrophilic, electrically neutral and do not bind proteins. Molecular sieving due to gravity-induced compression of the gel can be excluded for same reasons. In a medium without molecular sieving properties a high-resolution can be expected.
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. It is essentially non-sieving and optimal for electrophoresis of proteins that are smaller and larger than 200 ku (cf. agarose gel electrophoresis). Proteins migrate in it more or less on the basis of their free mobility. For these reasons interactions of the gel with the biomolecules are negligibly low and the proteins separate cleanly and predictably (see figure Electropherogram). The separated metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides, metallothionein, phytochelatins) are not dissociated into apoproteins and metal cofactors.
Reproducibility and recovery
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 (see figure Electropherogram). A shifting peak in the respective electropherogram may indicate 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, whereas exceeding this time limit (> 69 hr) is an indicator of gel aging. The phenomenon of gel aging is closely connected to long-term viscosity decrease of aqueous polyacrylamide solutions and increased swelling of hydrogels. In these cases the electroelution and detection of proteins is not reproducible (see figures Electropherogram and Hydrolysis, cf. section Principle).
Under standard conditions excluding denaturing pre-analytical steps, metalloproteins with different molecular mass ranges and isoelectric points have been recovered in biologically active form at a quantitative yield of more than 95%. By preparative SDS polyacrylamide gel electrophoresis 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%. Preparative isotachophoresis (ITP) is applied for isolating palladium-containing proteins with molecular masses of 362 ku (recovery: 67%) and 158 ku (recovery: 97%).
Quantification and identification
Low 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) or total reflection X-ray fluorescence (TXRF), 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. Another established high sensitive detection method for the determination of (trace) elements is graphite furnace atomic absorption spectrometry (GF-AAS) (see figure Electropherogram). 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 analytes can be elucidated quantitatively by using solution NMR spectroscopy under non-denaturing conditions.
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 or Amyotrophic Lateral Sclerosis (ALS). Active CCS or SOD molecules contribute to intracellular homeostatic control of essential metal ions (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. Otherwise, free (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. The loss of (active) CCS increases the amyloid-β production in neurons which, in turn, is a major pathological hallmark of AD. Therefore, copper chaperone for superoxide dismutase is proposed to be one of the most promising biomarkers of Cu toxicity in these diseases. 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.
In the 20th century it was generally accepted that APS/TEMED-initiated reactions should be allowed to proceed for 5-15 min to approx. 1-2 hr to ensure maximum reproducibility in gel pore size of PAGE gels. In another review it is recommended to allow the gel to polymerize overnight at room temperature. Longer incubation times (16 hr to 2 wk) to finish the three-dimensional matrix (network) formation might not have any essential effects on the protein separation process. Westermeier annotates that due to silent polymerization the gels might be placed in a refrigerator shortly after (visible) polymerization. In the cold room, however, the (exothermic) reaction is not completed within a reasonable period of time. In a technical note ("Acrylamide Polymerization - A Practical Approach") Bio-Rad claims that polymerization may be largely complete after about 90 min at room temperature.
Now here is the question to answer: Is there only an approximate polymerization time in the range of minutes to hours, or are there reliable experimental data indicating the general validity of a specific polymerization time for any PAGE gels?
Progressively hydrolyzing into polyacrylic acid and ammonia polyacrylamide gels were further considered as inherently unstable with respect to polymer resistance to alkaline media (see figure Hydrolysis). However, this hypothesis cannot be generalized. As acrylamide starts to hydrolyze at pH around 10 the hydrolysis rate of aqueous solutions of polyacrylamide is at a maximum at pH 4, and at a minimum at pH 10. Slow hydrolytic processes of fresh gels (pH 10.00) polymerized for 69 hr do not affect the peak-elution times of proteins in the electropherogram (see figure Electropherogram). Upon physiological conditions (pH 8.00) and after overnight polymerization, however, only semi-quantitative analysis of low amounts of the cofactor of interest could be performed by native PAGE using GF-AAS as detection method. Therefore, the relationship between the polymerization time, gel stability (incomplete polymerization, hydrolysis), pH and analytical results had to be investigated addressing the absolute quantification of specific protein molecules:
In 2001 a new basic principle of gel electrophoresis was discovered at the Forschungszentrum Jülich, applied for patent in 2003 and subsequently issued in 2014. This invention provided the first conclusive evidence that the time of polymerization of a polyacrylamide gel directly affects the result of protein purification because the separation properties implying the mechanical and chemical stability of a gel and its pores are determined by this parameter. As a consequence, the peak-elution times of the separated metalloproteins in the electropherogram may vary considerably (see figure Electropherogram). On the other hand, the results of protein electrophoresis can be optimized in terms of reliability by strict adherence to a standardized time of polymerization (69 hr). This optimization process of selected electrophoretic parameters paved the way to "quantitative native PAGE" implying high reproducibility in gel pore size and high-yield electroelution of proteins in biological samples.
Although the quality, quantity and mixing ratio of TEMED, APS and AA/Bis-AA as well as the ambient temperature are the most important factors to initiate and impel the polymerization reaction of PAGE gels, it is evident that the time of polymerization is the limiting factor in these physico-chemical processes to form an inherently stable network of polymers. Systematic investigations of the hydrogel stability over time reveal significant changes in gel structure (porosity) by day 3 (72 hr) after (visible) polymerization of 10% T, 15% T, and 20% T gels (under acidic conditions). This study is supported by another systematic investigation on gel aging, indicating that low concentrations of carboxyls are detectable after 3 days of acrylamide polymerization.
These results are in excellent agreement with the finding that the aging of fresh polyacrylamide gels begins after 69 hr cross-linking and gelation time revealing a shift of the peak-elution times of proteins due to gel swelling induced by hydrolysis of carboxamide groups into carboxylate anions (see figures Electropherogram and Hydrolysis). Thus, the polymerization time of 69 hr is a pH-independent stability constant in the pH range of about 4 (acrylamide/aqueous solution) to 10 (acrylamide/buffer solution) for acrylamide gels with total monomer concentrations in the range of 4 to 20% T.
First publications (2009) concerning the medical applications of this technique were edited or co-authored by the Münsteran human geneticist Prof. em. Jürgen Horst and the well-known American scientist and internationally recognized expert in the fields of DNA sequence analysis and protein electrophoresis David E. Garfin (see figure David Garfin). Beginning in the late 1960s D.E.G. used paper electrophoresis to sequence oligonucleotides prepared from tobacco mosaic virus ribonucleic acid. In the 1970s and the early 1980s Dr. Garfin became one of the most important investigators and early pioneers in the field of prion research (scrapie) in the team of the later Nobel Prize winner Stanley B. Prusiner at the UCSF.
In addition to his pioneering work on one- and two-dimensional gel electrophoresis in the following decades at Bio-Rad Laboratories as Proteomics Applications Manager he became co-editor of the Handbook of Isoelectric Focusing and Proteomics (2005) and co-authored a worth reading chapter on bioseparation methods in the Kirk-Othmer Encyclopedia of Chemical Technology (2007). Furthermore, Garfin is author of several breakthrough articles part of which are cited here and which also substantially complement and confirm the method presented here. For significant contributions to electrophoresis in both the engineering and biology communities Dave Garfin received the 2013 AES Electrophoresis Society Career Award in San Francisco (see figure David Garfin).
Garfin, Horst and some researchers from the Forschungszentrum Jülich anticipated that the above-mentioned approach might be implemented in the therapy and diagnosis of several protein-misfolding diseases: on the one hand, copper chaperone for superoxide dismutase may serve as a fluid-based biomarker for Cu toxicity in neurodegenerative diseases (cf. section Applications), on the other hand, copper chaperones, and smaller non-proteinogenic species (e.g., Cu orotate, Li chloride) are indicated as lead compounds for the etiological treatment of Alzheimer's disease.
According to the Hofmeister series salt effects could be one approach for the treatment of Alzheimer's disease by using certain salts for dissolving protein aggregates or inhibiting amyloid formation. Adding salts to complex protein mixtures, however, may induce a shift of isoelectric points, and thus, affect protein structure (shape) and activity. In this context, one study found that kosmotropic salts (e.g., ammonium sulfate) that were used for the precipitation of native proteins in solution, subsequently, may result in denatured high molecular weight metalloproteins in the same solution. Ions which promote aggregation or prefractionation in proteome analysis, simultaneously cause denaturation of the native conformation of the proteins of interest. In opposite case chaotropic salts force globular proteins to unfold. For these reasons, restoring and maintaining the physiological states of aggregated and/or unfolded (metal) proteins by applying Hofmeister salts in the therapy of Alzheimer's disease is very unlikely to happen. For example, Li chloride caused a reduction in protein synthesis and hence the level of amyloid-β peptides, however, this compound may also generate severe side effects induced by long-term, high-dose lithium.
Curcumin is one of the most promising therapeutic agents for inflammation, cystic fibrosis, Alzheimer's disease and cancer because these biomolecules scavenge radicals and maintain the levels of (active) antioxidant enzymes (e.g., SOD1) in the presence of copper. Curcumin is almost free from side effects, however, limited for application due to its poor bioavailability. Combining modern biochemical techniques and plant phenotyping platforms may help to provide bioactive therapeutic proteins as a major basis for pharmacological efficiency (non-toxic, high specificity, well known mechanisms of action) and bioeconomy approaches (high-throughput screening) in conformational diseases.
This preparative and analytical technique is based on a new principle and a new constant of acrylamide gel electrophoresis implying the accurate control of gel pore size and stability by the time of polymerization of acrylamide. Polyacrylamide gels are inherently stable at 69 hr of polymerization time. The electroelution and detection of proteins in reproducible and quantitative amounts are major features of reliable electrophoretic separation systems. As both low and high molecular weight (metal) proteins and their respective structures can be analysed in a single run, a combined procedure of solution NMR, QPNC-PAGE, and ICP-MS may be key for the diagnosis and therapy of several protein-misfolding diseases related to dyshomeostasis of biometal metabolism in the human brain. Principally biological proteins possess the pharmacological potential to restore and maintain the homeostasis of trace transition metal ions in physiological and pathophysiological conditions.
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- Chapters on 1-D / 2-D Gel Electrophoresis, and Isoelectric Focusing are provided by AES Electrophoresis Society Learning Center: Foci