Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS‐PAGE and 2D gel electrophoresis

Highly homogenous α zein protein was isolated from maize kernels in an environment‐friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI‐TOF‐MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS‐PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS‐PAGE bands matched to 30 amino acid sequence entries out of 102 non‐redundant data base entries. MALDI‐TOF‐MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in‐gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in‐gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.     

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.     

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.     

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.     

Albumin‐bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS‐PAGE, thiols were freed from protein with tri‐n‐butylphosphine and successively derivatized with 5‐iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 μg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.     

A competent extraction method of plant proteins for 2-D gel electrophoresis

The efficient extraction of high‐quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea‐/thiourea‐ and NP‐40‐containing solution. Protein profiles were examined on pH 3–11 non‐linear IEF strips and SDS‐PAGE and compared with extracts using the established method of acetone‐10% TCA/0.07% 2‐mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1–8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2‐mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250–150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25–15 kDa. The MALDI‐TOF‐MS of differential spots from acetone containing 10% TCA/0.07% 2‐mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.     

Alpha‐1 antitrypsin variants in plasma from HIV‐infected patients revealed by proteomic and glycoproteomic analysis

Novel tools are necessary to explore proteins related to human immunodeficiency virus (HIV) infection. In this work, proteomic and glycoproteomic technology were employed to examine plasma samples from HIV‐positive patients. Through comparative proteome analysis of normal and HIV‐positive plasma samples, 19 differentially expressed protein spots related to 12 non‐redundant proteins were identified by ESI‐ion trap MS. Among these, the 130‐kDa isoform of α‐1‐antitrypsin was found to be decreased in HIV‐positive patients while another variant with a molecular weight of 40 kDa was increased. SWISS‐2‐D‐PAGE reference gel and protein sequence comparisons of the 40‐kDa protein showed homology with α‐1‐antitrypsin minus the N‐terminus, and its identity was further confirmed by 1‐D Western blotting and glycoproteomic analysis. In all, our results showed that proteomics and glycoproteomics are powerful tools for discovering proteins related to HIV infection. Furthermore, this 40‐kDa variant of α‐1‐antitrypsin found in the plasma of HIV‐positive individuals may prove to be a potentially useful biomarker for anti‐HIV research according to bioinformatics analysis.     

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris‐acetate polyacrylamide gels

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.     

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.     

BOP: A basic phenylalanine‐rich oligo‐peptide located on the surface of glycolate oxidase influences its pI values

Glycolate oxidase (GO) consists of identical subunits and therefore should show one definite pI value, but the isolated GO exhibited a range of pIs. This study investigated the underlying cause of this phenomenon. GO was purified and showed a molecular weight of 40 kDa by SDS‐PAGE. Elution behavior on DEAE‐cellulose chromatography and cellulose acetate membrane electrophoresis indicated that the purified GO was highly basic (pI>10.0). Repeated IEF and cIEF analysis showed that the pI of the purified GO was in the range of 10.0–3.25, either in a smear form or as distinct bands. 2‐DE analysis showed that the 40 kDa subunit of GO displayed variable pIs from 9.6 to 3.65. It was likely that the purified GO was actually a complex consisted of GO and an unknown protein. CE‐SDS, SDS‐cellulose acetate membrane electrophoresis and amino acid compositions indicated that the unknown protein was a highly basic polymer (BP) consisting of basic and phenylalanine‐rich oligo‐peptide (BOP). Many BOPs are located on the surface of the acidic GO via ionic and hydrophobic interactions and formed GO‐BOP complex (GC), resulting in a highly basic GC although GO itself was acidic. This hypothesis was further supported by the facts that anti‐GC serum failed to recognize GO, and GC showed a peak at 257 nm although GO has few phenylalanine residues. Irregular and incomplete disassociation between GO and BOP was observed in IEF and cIEF, resulting in various intermediates with different ratios of GO/BOP, which could be the reason for the range of pIs observed for GO.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.     

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.     

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.     

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.     

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.     

Comparison of the Electrochemical Behavior of the High Molecular Mass Cadmium Proteins in Arabidopsis thaliana and in Vegetable Plants on Using Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC‐PAGE)

In Arabidopsis cytosol (supernatant) and in supernatants of vegetable plants high molecular mass cadmium proteins with molecular mass 200 kDa were isolated by using p reparative n ative c ontinuous p olya crylamide g el e lectrophoresis (PNC‐PAGE). Because of a different electrochemical behavior of the Cd proteins in Arabidopsis and endive supernatants on using the same PAGE method, it is concluded that the high molecular mass cadmium proteins of Arabidopsis and endive possess different isoelectric points. Consequently, different chemical structures of the Cd proteins with molecular mass 200 kDa are present in Arabidopsis thaliana and in endive. During the electrophoretic separation of vegetable metalloproteins by using the Model 491 Prep Cell from BioRad, electroanalytical processes like electrode reactions may play an important role.     

Poly (L‐lactide)‐Degrading Enzyme Produced by Amycolatopsis sp.

Poly (L ‐lactide) (PLLA)‐degrading enzyme was produced in a liquid culture of Amycolatopsis sp. (strain 41). In comparison with polyester substrates, silk powder from silkworm cocoons was the most effective in inducing enzyme production within 5 d. Application to DEAE and Superdex 75 columns resulted in a major protein with molecular weight estimated to be 42 kDa from size exclusion chromatography or 40 kDa from SDS‐PAGE analysis. Optimum pH and temperature are 6.0 and 37–45°C, respectively. Besides PLLA, the enzyme degrades casein, silk powder and Suc‐(Ala)₃‐pNA at an even lower level than Proteinase‐K, but not Suc‐(Gly)₃‐pNA, poly (ε‐caprolactone) and poly (β‐hydroxybutyrate).     

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli

An efficient affinity‐purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4‐Aminopyrrole‐2‐carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C₈ column and reducing SDS‐PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS‐PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ～46 kDa. The desorption constant K_d and theoretical maximum absorption Q_max were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.     

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.