Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

In‐gel equilibration for improved protein retention in 2DE‐based proteomic workflows

The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second‐dimension SDS‐PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in‐solution‐based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second‐dimension SDS‐PAGE gel, referred to as in‐gel equilibration. We show that in‐gel equilibration is effective at reduction and alkylation in SDS‐PAGE gels. Quantification of whole‐cell extracts separated on 2DE gels shows that in‐gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.     

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2′‐(1E,1′E)‐2,2′‐(4‐(dicyanomethylene)‐4H‐pyrane‐2,6‐diyl)bis(ethene‐2,1‐diyl)bis(sodium benzenesulfonate) salt ( 1 ), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high‐throughput SDS‐PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in‐gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60–90 min under optimum conditions much shorter than that required by the less‐sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in‐gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1 , which is a modified protocol of blue native PAGE (BN‐PAGE). Thus, 1 may facilitate high‐sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

A novel polyacrylamide gel system for proteomic use offering controllable pore expansion by crosslinker cleavage

SDS‐PAGE is still one of the most widespread separation techniques in proteomic research and usually coupled to subsequent MS measurement for protein identification. The proteins are digested while embedded in the gel matrix. The resultant peptides are eluted out of the gel and finally analyzed. The in‐gel digestion process suffers from several drawbacks which influence the experimental outcome with respect to protein sequence coverage and detection sensitivity. Limited accessibility of the protease to the substrate protein and insufficient peptide extraction represent the two major problems. To specifically target these issues, we established a novel partly reversible gel system, in which the gel matrix can be conditionally cleaved to increase the pore diameters. By using a crosslinker mixture consisting of Bis and ethylene‐glycol‐diacrylate the acrylamide filament interconnections can be partly hydrolyzed in alkaline solution. The new hybrid gels have been tested to be compatible with a variety of acidic staining techniques. They exhibit similar electrophoretic performance compared with regular solely Bis‐based gels, but yield significantly better MS results. Thus, the Bis/ethylene‐glycol‐diacrylate SDS‐PAGE gel system is a promising alternative for MS‐based in‐gel workflows and might be transferred to other gel‐electrophoretic applications.     

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications

Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution-phase isoelectric focusing is an effective alternative to sucrose-gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence-based multiplexing dye technologies greatly extend the capacity of SDS-polyacrylamide gel electrophoresis with respect to the investigation of proteome-wide changes in protein expression and post-translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two-dimensional gel electrophoresis.     

Urine protein quantification in stacking gel by SDS‐PAGE

Urine total protein concentration is usually measured by the pyrogallol red‐molybdate (PRM) assay in clinical laboratories, but it is often subject to sample interference. Here, we introduce a stacking gel‐based method for accurate protein quantitation. In this method, the urine protein samples are run into the stacking gel by SDS‐PAGE where it is concentrated into a single band, and then quickly stained by 0.001% Coomassie at high temperature. High correlations were found between the BSA and urine protein standards (R² = 0.997 and 0.990, respectively). Addition of 80 ng urine protein standard into each of the ten clinical urine specimens with questionable PRM results yielded the expected increase in the results by this method. Comparison of the PRM method and with the gel quantitation approach on about 60 clinical urine samples demonstrated a general consistency between the results (R² = 0.825), but in PRM samples with lower protein concentration showed more variations. Overall, the stacking gel method might be a good alternative for clinical urine samples with suspicious protein concentration results.     

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Staining of proteins for 2D SDS‐PAGE using Coomassie Blue—speed versus sensitivity?

Several new fast staining protocols for the visualization of proteins separated by SDS‐PAGE utilizing Coomassie Blue staining (CBS) have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS‐PAGE of serially diluted protein samples. However, this approach is not predictive and satisfactory for 2D SDS‐PAGE capable of resolving hundreds or thousands of different proteins in a single analysis. In this work, a new fast staining protocol recently introduced by Dong et al. (PLoS One 2011, 6, e22394) was compared to colloidal CBS. The number of detectable spots in 2D SDS‐PAGE of identical blood plasma samples in repeated runs was chosen as a sensitivity criterion. Further, the influence of gel boiling on the subsequent protein identification by MS was investigated. In spite of its advantages, the staining protocol according to Dong et al. (PLoS One 2011, 6, e22394) seems to be less sensitive than colloidal Coomassie staining when the number of detected spots is the evaluating criterion. No obvious influence of gel boiling on the protein identification was observed.     

Property and Application of BACy‐Based Functional Hydrogels

Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acrylamide and N,N′‐methylenebisacrylamide (NMBA) have been frequently used in the biochemical technique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to resolve protein mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross‐linking of acrylamide and N,N′‐bisacrylylcystamine (BACy). In addition, we have compared the BACy‐based hydrogel with the NMBA‐based polyacrylamide hydrogel for their physical properties such as swelling ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether BACy‐based polyacrylamide hydrogel could be applied to SDS‐PAGE and proteomics research. The results showed that this type of hydrogel is capable of separating proteins and facilitates further in‐gel protein digestion and the following protein identifications by mass spectrometry. In summary, our study provides a basis for the putative application of BACy‐based hydrogels.     

Performance of nondenaturing micro 2-DE followed by third-dimension SDS-PAGE in the analysis of Escherichia coli soluble proteins

In a previous paper, we reported on the analysis of Escherichia coli (strain K‐12) soluble proteins by nondenaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF [Manabe, T., Jin, Y., Electrophoresis 2010, 31, 2740–2748]. To evaluate the performance of the 2‐DE/3‐DE technique, a nondenaturing 2‐DE gel just after the second‐dimension run was cut into 12 vertical strips, each 2 mm‐wide strip was set on a micro slab gel, and third‐dimension SDS‐PAGE was run in parallel. Each of the twelve 3‐DE gels showed about 150–200 CBB‐stained spots. Two of the 3‐DE gels were selected for the assignment of polypeptides using MALDI‐MS‐PMF and totally 161 polypeptides were assigned on the two 3‐DE gels, in which 81 have been assigned on the nondenaturing micro 2‐DE gel and 80 were newly assigned. Most of the newly assigned polypeptides resided in faintly stained spots on the 3‐DE gels, which indicates that the polypeptides were purified in the process of the third‐dimension separation. The comparisons of the apparent mass values estimated from the second‐dimension (nondenaturing pore‐gradient PAGE) mobility with those estimated from the third‐dimension (SDS‐PAGE) mobility suggested the oligomer structures of the assigned polypeptides and they matched well with those described in a database (UniProtKnowledgebase). The technique of nondenaturing micro 2‐DE/3‐DE, combined with MALDI‐MS‐PMF, could become an efficient method to obtain information on the quaternary structures of hundreds of cellular soluble proteins simultaneously because of its high efficiency in protein/polypeptide separation and assignment.     

Comparison of antimicrobial peptide purification via free‐flow electrophoresis and gel filtration chromatography

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad‐spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free‐flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS‐PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30‐fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2‐fold dilution but the latter had ∼13‐fold dilution. Furthermore, Tricine‐SDS‐PAGE, Native‐PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.     

Cover Picture: Cyclometalated Platinum(II) Complexes as Highly Sensitive Luminescent Switch‐On Probes for Practical Application in Protein Staining and Cell Imaging (Chem. Eur. J. 15/2009)

Photoluminescent cyclometalated platinum(II) complexes can bind non‐covalently and non‐specifically towards various proteins and such bonding is accompanied by a dramatic increase in the intensity of photoluminescence in the visible spectral region. It can be practically applied for staining protein mixtures in 1D and 2D SDS‐PAGE, giving emissive gel images directly under UV light without any de‐staining procedures. For more information see the Communication by C.‐M. Che et al. on page 3652 ff. (SDS‐PAGE=sodium dodecyl sulfate polyarcylamide gel electrophoresis).

     

Combination of SDS‐PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time‐consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS‐PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS‐PAGE were recovered from gel by treatment with SDC‐containing buffer. Usage of SDC‐containing buffer as extraction solvent and ethanol‐based staining solution enhanced the recovery of intact IgG from SDS‐PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N ‐glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.     

Detection of Chitinolytic Enzymes in Ipomoea Batatas Leaf Extract by Activity Staining after Gel Electrophoresis

A non‐denatured SDS‐PAGE followed by in‐gel activity staining using embedded glycol chitin as a substrate was used to identify the proteins with chitinolyitc activities from sweet potato leaf extract. At least two chitinase activity zones can be clearly identified on the gel at positions with estimated molecular weights of 54.1～55.6 kDa and 39.6 kDa. Furthermore, our data also indicate that the activity of the larger one can withstand the standard SDS‐PAGE sample preparation. Both of these chitinases, however, are different from that of the previously identified chitinase in sweet potato leaves, which has a molecular weight of 16 kDa. By using an embedded substrate, our method has superior sensitivity in detecting chitinases with higher molecular weights. It is a simple, affordable way and may aid in the future discovery of new chitinases.     

Polyacrylamide Gel Electrophoresis of Insulin

Abstract

Four kinds of polyacrylamide gel electrophoresis (PAGE) were applied to insulin and peroxynitrite‐treated insulin. The Native‐PAGE had a better resolution than sodium dodecyl sulfate (SDS)‐PAGE, SDS‐urea‐PAGE, and even Tricine‐SDS‐PAGE. Reduction and nonreduction of insulin and peroxynitrite‐treated insulin in Native‐PAGE showed that four tyrosine residues in insulin molecular could be nitrated by peroxynitrite and that alkylation with iodoacetamide was better than no alkylation and alkylation with iodoacetic acid, which would introduce negative charges to the peptides. The method of Native‐PAGE was suitable to analysis of insulin and its analogs, even other peptides of low molecular weight.     

Complex Formation between the Lumenal Domain of Adenovirus E3–19k Protein and the Extracellular Domain of Class I MHC Molecule In Vitro

The Ad2 E3–19k protein inhibits the transport of newly synthesized class I MHC molecules to the cell surface, thereby interfering with antigen presentation. The details of the interaction between E3–19k protein and class I MHC molecules have not been well‐defined. In this present study, we describe the use of gel filtration HPLC for confirming the binding interaction of two domain proteins, E3–19k and MHC class I antigen, and subsequently the characterization of protein complex by SDS‐PAGE. Our results demonstrate the complex formation between Ad2 lumenal E3–19k (108 amino acids, wt 108) and HLA‐A*0201 molecule in vitro. Titration experiments will be employed in the future to determine stoichiometry and verify the specific interactions.     

Observation of orientation and relaxation of protein-sodium dodecyl sulfate complexes during pulsed intermittent field polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) of proteins denatured with SDS (sodium dodecyl sulfate) has been used successfully to separate proteins according to their molecular mass. In spite of the extensive use of this technique, the motion of the protein-SDS complex in a polyacrylamide gel is still not understood. Here we report on the observation of the orientation (in the field direction) and relaxation of protein-SDS complexes during pulsed intermittent field PAGE experiments. The results give an indication of the stiffness of the molecules and may be useful for the development of a technique to improve the separation of large proteins using pulsed electric fields.     

A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid

A fluorescent quenching detection method for phosphoproteins in SDS‐PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al³⁺ in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA‐Al³⁺‐phosphoprotein appended complex. Approximately 4–8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS‐PAGE), which is similar to that of the most commonly used Pro‐Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC‐MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel‐separated phosphoproteins.