MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross‐linked polyacrylamide gels

Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross‐linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS‐PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom‐developed workflow. It includes protein extraction with an ammonia–SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI‐TOF analysis was implemented using methanol–chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as ‘real’ biological samples (8–75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI‐TOF MS, were in the range of 0.1 to 5 μg, and the required amount increased with increasing mass.     

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.     

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.     

Characterization of the pore structure of aqueous three-dimensional polyacrylamide gels with a novel cross-linker

The properties of polyacrylamide hydrogels synthesized with a novel hexafunctional (three double bonds) cross-linker, hexahydro-1,3,5-triacryloyl-s-triazine (1a), was evaluated and compared to the currently used tetrafunctional (two double bonds) cross-linker N,N-methylenebisacrylamide (Bis). A variety of characterization techniques that require very little sample preparation and data handling were chosen and include polymerization temperature profiles and conversions, water swelling, differential scanning calorimetry (DSC), polyacrylamide gel electrophoresis (PAGE), Gradiflow electrophoretic separation process and scanning electron microscopy (SEM). The alternative use of 1a compared to Bis results in polyacrylamide gels with larger pore sizes and a broad pore size distribution.     

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.     

Synthesis of gels by means of Michael addition reaction of multi-functional acetoacetate and diacrylate compounds and their application to ionic conductive gels

Michael-addition reactions of multi-functional acetoacetate, meso-erythritol tetraacetoacetate (ETAA), trimethylolpropane triacetoacetate (TPTAA), and diacrylate compounds, 1,4-butanediol diacrylate, 1,6-hexanediol diacrylate, 1,9-nonanediol diacrylate, or poly(ethylene glycol) diacrylate (PEGDA), in dimethyl sulfoxide have successfully yielded the corresponding gels in the presence of 1,8-diazabicyclo[5.5.0]undecane-7-ene as a catalyst at room temperature. The gel formation rates of the reaction systems with TPTAA were higher than those with ETAA. The gels prepared with the alkyl diacrylate compounds or low molecular weight PEGDA showed higher Young's modulus in compression test. The ETAA-PEGDA gels were also prepared in propylene carbonate containing Li ion or in an ionic liquid. These gels showed good ionic conductivity with conductivity value as high as 2.26 and 2.38 mS/cm at room temperature for the Li ion and ionic liquid containing systems respectively.     

Comparative study for separation of aquatic humic substances by capillary zone electrophoresis using uncoated, polymer coated and gel-filled capillaries

Several comparative capillary zone electrophoresis (CZE) experiments were carried out by means of uncoated, polyvinyl alcohol (PVA) and polyacrylamide (PAA) coated silica open tubular capillaries and gel-filled capillaries (linear non-cross-linked polyacrylamide, PAGE, by a pre-coated PAA capillary) using different kinds of background electrolytes (BGEs) and organic modifiers for characterization of aquatic dissolved humic matter (DHM). Organic compounds, such as acetic acid, acetate buffer, methanol, ethylene glycol, acetonitrile, dimethylsulphoxide, 5 M urea and sodium dodecyl sulphate (SDS) were tested as sample modifiers to improve the separative power. The fractionation mode by a PVA coated open tubular capillary using 40 mM phosphate buffer at pH 6.8 and 5 M urea-water as the sample modifier turned out to be fairly practical as well as its PAA homologue. Linear non-cross-linked PAGE with 10% gel concentration and 5 M urea-water as the sample modifier using 40 mM phosphate buffer at pH 6.8 produced the most reliable results as to the adaptation of physical gels, especially if the interactions of humic solutes with the gel matrix are not critical. The addition of SDS in the linear PAGE gel increased the interaction of humic solutes with the gel matrix but also improved the separative power and strengthened the chaotropic effect of the urea modifier.     

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.     

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.     

Hydrogels for in situ encapsulation of biomimetic membrane arrays

Hydrogels are hydrophilic, porous polymer networks that can absorb up to thousands of times their own weight in water. They have many potential applications, one of which is the encapsulation of freestanding black lipid membranes (BLMs) for novel separation technologies or biosensor applications. We investigated gels for in situ encapsulation of multiple BLMs formed across apertures in a hydrophobic ethylene tetrafluoroethylene (ETFE) support. The encapsulation gels consisted of networks of poly(ethylene glycol)‐dimethacrylate or poly(ethylene glycol)‐diacrylate polymerized using either a chemical initiator or a photoinitiator. The hydrogels were studied with regards to volumetric stability, porosity, and water permeability. All hydrogels had pore sizes around 7 nm with volumetric changes >2% upon crosslinking. Photoinitiated hydrogels had a lower hydraulic water permeability compared to chemically initiated hydrogels; however, for all hydrogels the permeability was several‐fold higher than the water permeability of conventional reverse osmosis (RO) membranes. Lifetimes of freestanding BLM arrays in gel precursor solutions were short compared to arrays formed in buffer. However, polymerizing (crosslinking) the gel stabilized the membranes and resulted in BLM arrays that remained intact for days. This is a substantial improvement over lifetimes for freestanding BLM arrays. Optical images of the membranes and single channel activity of incorporated gramicidin ion channels showed that the lipid membranes retained their integrity and functionality after encapsulation with hydrogel. Our results show that hydrogel encapsulation is a potential means to provide stability for biomimetic devices based on functional proteins reconstituted in biomimetic membrane arrays. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

The effect of sodium dodecyl sulfate and anion‐exchange silica gel on matrix‐assisted laser desorption/ionization mass spectrometric analysis of proteins

Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), this surfactant can often cause signal suppression. We have previously reported an on‐probe sample preparation method using a suspension of anion‐exchange silica gel and sinapinic acid (i.e., gel‐SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on‐probe sample preparation method using both SDS and the gel‐SA suspension improved the detection limit of protein samples in the MALDI‐MS analysis by about ten‐fold as compared to that of protein samples without SDS and the gel‐SA suspension. This method can be applied not only to the MALDI‐MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative Study of In Situ Loaded Antibody and PEG‐Fab NIPAAM Gels

Hydrogels can potentially prolong the release of a therapeutic protein, especially to treat blinding conditions. One challenge is to ensure that the protein and hydrogel are intimately mixed by better protein entanglement within the hydrogel. N‐isopropylacrylamide (NIPAAM) gels are optimized with poly(ethylene glycol) diacrylate (PEDGA) crosslinker in the presence of either bevacizumab or PEG conjugated ranibizumab (PEG₁₀‐Fab_rani). The release profiles of the hydrogels are evaluated using an outflow model of the eye, which is previously validated for human clearance of proteins. Release kinetics of in situ loaded bevacizumab‐NIPAAM gels displays a prolonged bimodal release profile in phosphate buffered saline compared to bevacizumab loaded into a preformed NIPAAM gel. Bevacizumab release in simulated vitreous from in situ loaded gels is similar to bevacizumab control indicating that diffusion through the vitreous rather than from the gel is rate limiting. Ranibizumab is site‐specifically PEGylated by disulfide rebridging conjugation. Prolonged and continuous release is observed with the in situ loaded PEG₁₀‐Fab_rani‐NIPAAM gels compared to PEG₁₀‐Fab_rani injection (control). Compared to an unmodified protein, there is better mixing due to PEG entanglement and compatibility of PEG₁₀‐Fab_rani within the NIPAAM‐PEDGA hydrogel. These encouraging results suggest that the extended release of PEGylated proteins in the vitreous can be achieved using injectable hydrogels.     

Synthesis and swelling behavior of thermosensitive hydrogels based on N-substituted acrylamides and sodium acrylate

A series of hydrogels based on N-isopropylacrylamide, sodium acrylate, and N-tert-butylacrylamide were synthesized by free radical polymerization in a mixture of dioxane and water with tetra(ethylene glycol) diacrylate as the crosslinker and benzoyl peroxide as the initiator. The swelling behavior including the swelling rate of the crosslinked gels in water was studied with gravimetric method. The swelling ratio of the gel (0.1 mol% crosslinking) can reach 420 g/g at 20 °C and such a gel can release 96% of the water absorbed at 40 °C. The lower critical swelling temperature (LCST) of the copolymers can be adjusted by changing the chemical composition of the polymers. Such crosslinked gels can be potentially used as thermosensitive superabsorbent because of their high water uptake and thermal sensitivity.     

Differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG‐conjugated proteins. Human growth hormone and alpha‐lactalbumin modified by linear or branched PEGs with molecular weight of 10 kDa were analyzed by SEC, MALDI‐TOF MS, SDS‐PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG‐proteins on SEC and MALDI‐TOF MS were small, but their electrophoretic behaviors on SDS‐PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG‐proteins, in which the branched PEG‐proteins exhibited a narrower peak and longer migration time than the linear PEG‐proteins. This phenomenon may explain the longer circulation half‐life for the branched PEG‐proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG‐proteins.     

Synthesis of Biodegradable Hydrogel by Radical Ring‐Opening Polymerization of 2‐Methylene‐1,3,6‐Trioxocane

2‐Methylene‐1,3,6‐trioxocane (MTC), maleic anhydride (MA), and poly(ethylene glycol diacrylate) 400 (PEGD) or divinyl adipate (DVA) were terpolymerized in the presence of a radical initiator. Though the obtained gels did not absorb deionized water, the gels could absorb deionized water and saline after hydrolyzing the MA carboxylate anhydride functionality. The absorbing ratios of deionized water and saline were high. The biodegradability of the hydrogels after hydrolysis was found to be good by a biochemical oxygen demand (BOD)‐tester using soil.     

The effect of the biological variability of samples on Coomassie blue dye based fast staining for SDS‐PAGE in nonfixed gels

Fast‐staining protocols based on the use of Coomassie blue dye for SDS‐PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast‐staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast‐staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS‐PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.     

The stability of semi-interpenetrating polymer networks based on sulfonated polyimide and poly(ethylene glycol) diacrylate for fuel cell applications

A series of the semi-interpenetrating polymer network (semi-IPN) membranes based on sulfonated polyimide and poly(ethylene glycol) diacrylate were prepared and characterized comparing with pure sulfonated polyimide membrane and commercially available membrane, Nafion^® 117. The proton conductivity increased with the increase of poly(ethylene glycol) diacrylate contents in spite of the decrease in ion exchange capacity which is a key factor to improve the proton conductivity. The water stability of semi-IPN membranes containing poly(ethylene glycol) diacrylate is higher than the pure sulfonated polyimide membrane. Morphological structure showed that amorphous nature of the films also increased with the poly(ethylene glycol) diacrylate contents, which could make a crosslink, so that the crystallinity of polyimide could disappear. Semi-IPN membranes based on sulfonated polyimide and poly(ethylene glycol) diacrylate, which show good conductivity comparable to Nafion^® 117 in the range of 20-50% content of poly(ethylene glycol) diacrylate, could be promising proton conducting membranes in fuel cell application.     

Electrophoresis of cereal storage proteins

Cereal proteins have been studied by a number of analytical techniques over the years. One of the major methodologies utilized by cereal chemists has been electrophoresis. Starting with moving boundary electrophoresis and progressing to slab gels and high-performance capillary electrophoresis, innovative methods have been developed to provide high resolution separations of difficult to separate proteins. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), acid-PAGE, isoelectric focusing, free zone CE, and even high-resolution two-dimensional HPLC-HPCE methods have been developed to separate cereal proteins. This review focuses on electrophoretic methods for separating and characterizing cereal storage proteins.