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.     

Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein

SDS‐PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein‐containing conjugates, mostly pegylated proteins. PEG‐maleimide (MAL) is frequently used to site‐specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C–S linkage between protein and PEG‐MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein was observed. It is a thiol‐independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG‐MAL modified conjugates although PEG‐vinylsulfone conjugates to thiol‐containing proteins also through a C–S linkage. Cleavage kinetics of PEG40k‐MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide‐thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG‐MAL modified protein/peptide by SDS‐PAGE analysis.     

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.     

Improved disc SDS‐PAGE for extraction of low molecular weight proteins from serum

The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS‐PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30 kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS‐PAGE and MALDI‐TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28 kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30 kDa. The experimental results obtained by plate SDS‐PAGE indicated the repeatability was satisfactory.     

Simplified method for concentration of mitochondrial membrane protein complexes

Extracting and concentrating mitochondrial protein complexes from gel strips after blue native PAGE (BN‐PAGE) can be daunting tasks using the traditional methods, such as electroelution, passive diffusion and centrifugal concentration. We present a simplified gel electrophoresis method to concentrate mitochondrial protein complexes with excellent recovery rate. Mitochondrial complex I present in a long gel strip from BN‐PAGE can be easily concentrated into a 0.8 cm gel strip when a second BN‐PAGE is performed with a Y‐shaped gel and the addition of 0.01% n‐dodecyl β‐D ‐maltoside and 0.001% SDS in the cathode buffer. Once completed, the concentrated protein complex in the gel strip is ready for SDS‐PAGE or proteomic studies.     

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass‐based separations (e.g. SDS‐PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time‐course SDS depletion curves to an exponential model, we calculate SDS depletion half‐lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS‐containing proteins.     

Estimation of protein concentration at high sensitivity using SDS‐capillary gel electrophoresis‐laser induced fluorescence detection with 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde protein labeling

3‐(2‐furoyl)quinoline‐2‐carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS‐CGE with LIF detection (SDS‐CGE‐LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N‐termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS‐CGE‐LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS‐CGE‐LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS‐CGE‐UV and SDS‐PAGE‐Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC‐ESI‐QTOF‐MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence.     

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.     

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.     

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos‐tag SDS‐PAGE migration

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos‐tag SDS‐PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos‐tag gels, the separation was not due to phosphorylation. The N‐terminal 47–61 region of ClpX was responsible for producing multiple phosphorylation‐independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up‐shifted bands. By systematic Ala‐scanning mutation analysis in the N‐47–61 region, we concluded that the Glu‐51 or Glu‐54 residue was responsible for the appearance of exaggerated mobility‐shifting bands. Histone H2A showed a much slower migration in Phos‐tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu‐62 or Glu‐65 residue caused the retarded migration. In addition, Phos‐tag SDS‐PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos‐tag SDS‐PAGE is induced by interactions between the Phos‐tag molecule and the carboxylate group of a specific Glu residue on the target.     

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS‐PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA‐F8 (anti‐MHC slow/I), SC‐71 (anti‐MHC 2a and 2x), 10F5 (anti‐MHC 2b), and BF‐35 (anti‐MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS‐PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS‐PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.     

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.     

Probing the limits of liquid droplet laser desorption mass spectrometry in the analysis of oligonucleotides and nucleic acids

In the present work we demonstrate the advantages of LILBID mass spectrometry (laser‐induced liquid bead ion desorption) in the analysis of nucleic acids and large oligonucleotides. For established methods like matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), the mass analysis of oligonucleotides or of noncovalent oligonucleotide‐protein complexes, in particular of very large ones, still represents a considerable challenge either due to the lack of native solutions or nonspecific adduct formation or due to a reduced salt tolerance or a high charge state of the ions. With LILBID, oligonucleotides, solvated in micro‐droplets of aqueous buffer at certain pH and ion strength, are brought into the gas phase by laser ablation. We show that our method is able to detect single‐ and double‐stranded oligonucleotides with high softness, demonstrated by the buffer dependence of the melting of a duplex. The absolute sensitivity is in the attomole range concomitant with a total analyte consumption in the femtomole region. The upper mass limit of oligonucleotides still detected with good signal‐to‐noise ratio with LILBID is the 1.66 MDa plasmid pUC19. With DNA ladders from short duplexes with sticky ends, we show that LILBID correctly reflects the relative thermodynamic stabilities of the ladders. Moreover, as an example for a specific DNA–protein complex we show that a NF‐κB p50 homodimer binds sequence specifically to its match DNA. In summary we demonstrate that LILBID, although presently performed only with low mass resolution, due to these advantages, is an alternative mass spectrometric method for the analysis of oligonucleotides in general and of specific noncovalent nucleic acid–protein complexes in particular. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

In‐capillary approach to eliminate SDS interferences in antibody analysis by capillary electrophoresis coupled to mass spectrometry

Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE‐MS method for the analysis of mAbs is presented analyzing SDS‐complexed samples. To obtain narrow and intensive peaks of SDS‐treated antibodies, an in‐capillary strategy was developed based on the co‐injection of positively charged surfactants and methanol as organic solvent. For samples containing 0.2% (v/v) of SDS, recovered MS peak intensities up to 97 and 95% were achieved using cetyltrimethylammonium bromide or benzalkonium chloride, respectively. Successful removal of SDS was shown in neutral coated capillaries but also in a capillary with a positively charged coating applying reversed polarity. The usefulness of this in‐capillary strategy was demonstrated also for other proteins and for antibodies dissolved in up to 10% v/v SDS solution, and in other SDS‐containing matrices, including the sieving matrix used in a standard CE‐SDS method and gel‐buffers applied in SDS‐PAGE methods. The developed CE‐MS approaches enable fast and reproducible characterization of SDS‐complexed antibodies.     

Studies on Aggregation Interaction between Reduced Denatured Egg White Lysozymes during Refolding Procedure in Urea Solution

The aggregation interaction between reduced-denatured egg white lysozymes during refolding procedure in urea solution was studied by means of reducing and non-reducing protein electrophoreses. Results of non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis （SDS-PAGE） of the supernatant and aggregate precipitate formed in refolding process show that except being refolded to native egg white lysozymes, the reduced-denatured lysozymes can also form the aggregates with molecular weights （MW） being separately about 30.0 and 35.0 kD, while the reducing SDS-PAGE and the refolding results in the presence of sodium dodecyl sulphate show that these aggregates are formed chiefly through the misconnection of disulfide bonds between the reduced-denatured lysozymes, and the aggregate precipitates are formed through the non-covalent interactions between the aggregates with molecular weight being about 30.0 kD. From the results of electrophoresis and size-exclusion chromatographic analyses, it can be inferred that the aggregates with molecular weights being about 30.0 and 35.0 kD are bi-molecular and tri-molecular egg white lysozyme aggregates, respectively. And finally, a suggested refolding mechanism of reduced-denatured egg white lysozymes in urea solution was presented.     

Studies of keratins in tongue coating samples of hepatitis B patients by mass spectrometry

Pooled tongue coating samples from 64 hepatitis B patients and 24 healthy adults were studied and a major band of differential proteins was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The differential proteins in this band were identified and proved to be keratins by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and Western blot analysis. Furthermore, relative quantification of the identified keratins was performed via using stable isotopic labeling and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), showing the higher expression level of these keratins in tongue coating samples of hepatitis B patients than healthy adults. These results provided additional information to understand the medical diagnosis depending on the tongue coating. Copyright © 2009 John Wiley & Sons, Ltd.     

Maize pollen enzymes after two-dimensional polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate

Twelve enzymes from mature pollen grains of maize were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2-D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic-oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1-D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2-D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed.