New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.     

Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles

Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH 8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50 cm bare fused-silica capillary was employed for obtaining adequate separations in about 12 min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples.     

Transgenic Soybean Seed as Protein Expression System: Aqueous Extraction of Recombinant β-Glucuronidase

Soybean is one of the plant species with potential to be used as seed-based bioreactor. As part of the downstream processing (DSP) of this technology, extraction is a key step, since it defines the composition of the solution from which the recombinant product will be purified. In the present work, the characteristics of soybean seeds used as a bioreactor were evaluated from a process engineering standpoint through analysis of the influence of pH and ionic strength on the extraction of recombinant β-glucuronidase (rGUS). Concentrations of recombinant protein and native soybean compounds were analyzed and compared with similar data from extraction studies using transgenic corn seeds as bioreactor. Efficient rGUS extraction was obtained at pH of around 5.5 with no addition of salt. Soybean seed extracts had lower levels of co-extracted native compounds, than corn seed extracts, and should be considered as a potential plant bioreactor in terms of DSP.     

In-gel protein N- and C-termini identification and its application for transgenic protein characterization

A new method for the determination of N- and C-termini of a protein isolated in a polyacrylamide gel is introduced. In-gel partial protein hydrolysis by hydrochloric acid is used to generate N- and C-terminal peptides for identification. This new method is complementary to existing techniques. The application of the in-gel protein termini identification technique to the characterization of the transgenic protein diacylglycerol acyltransferase (UrDGAT2A) purified from soybean seeds is also reported here. Both N- and C-termini of UrDGAT2A were successfully identified and the N-terminus was found to be blocked by acetylation. The analysis results of UrDGAT2A and two commercial proteins (bovine serum albumin (BSA) and alcohol dehydrogenase) are used to demonstrate the effectiveness of the method in identifying actual N- and C-termini, terminal truncation and blocking.     

Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.     

Homogeneous sample preparation for automated high throughput analysis with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

This work presents a simple method for obtaining homogeneous sample surfaces in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. The sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystals act as seeds for the new sample containing crystals which become much smaller in size and more evenly distributed than with conventional methods. This 'seed-layer' method was developed, optimised and compared with the dried-droplet method using peptides and proteins in the 1000-20,000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproducibility and sample washability as compared with the commonly used dried-droplet method. This methodology is applicable to alpha-cyanohydroxycinnamic acid, sinapinic acid and ferulic acid, which all form homogeneous crystal surfaces. Within-spot variation and between-spot variation was investigated using statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra. More than 90% of the sample locations results in high intensity spectra with relatively low standard deviations (RSDs). Typically obtained data showed an RSD of 19-35% within a sample spot as well as in-between spots for proteins, and an RSD of < or = 50% for peptides. Linear calibration curves were obtained within one order of magnitude using internal calibration with a point-RSD of 3% (n = 10). The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual sample within 15 sec, whereby a 100 spot target plate can be run in 25 min. High density target plates using the seed-layer method were prepared by spotting approximately 100 picoliter droplets onto the target, resulting in sample spots < or = 500 microns in diameter using a flow-through piezo-electric micro-dispenser. By using this automated sample preparation step lower standard deviations are obtained in comparison to manually prepared samples.     

Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low‐abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5‐enolpyruvylshikimate‐3‐phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography‐multiple reaction monitoring tandem MS (nano LC‐MS/MS‐MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)‐immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine‐containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A‐sepharose was used for the affinity capture of β‐conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable‐isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time‐consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.     

Measurement uncertainty from physical sample preparation: estimation including systematic error

A methodology is proposed, which employs duplicated primary sampling and subsequent duplicated physical preparation coupled with duplicated chemical analyses. Sample preparation duplicates should be prepared under conditions that represent normal variability in routine laboratory practice. The proposed methodology requires duplicated chemical analysis on a minimum of two of the sample preparation duplicates. Data produced from the hierarchical design is treated with robust analysis of variance (ANOVA) to generate uncertainty estimates, as standard uncertainties ('u' expressed as standard deviation), for primary sampling (ssamp), physical sample preparation (sprep) and chemical analysis (sanal). The ANOVA results allow the contribution of the sample preparation process to the overall uncertainty to be assessed. This methodology has been applied for the first time to a case study of pesticide residues in retail strawberry samples. Duplicated sample preparation was performed under ambient conditions on two consecutive days. Multi-residue analysis (quantification by GC-MS) was undertaken for a range of incurred pesticide residues including those suspected of being susceptible to loss during sample preparation procedures. Sampling and analytical uncertainties dominated at low analyte concentrations. The sample preparation process contributed up to 20% to the total variability and had a relative uncertainty (Uprep%) of up to 66% (for bupirimate at 95% confidence). Estimates of systematic errors during physical sample preparation were also made using spike recovery experiments. Four options for the estimation of measurement uncertainty are discussed, which both include and exclude systematic error arising from sample preparation and chemical analysis. A holistic approach to the combination and subsequent expression of uncertainty is advised.     

Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract

Tobacco has been studied as a possible host for the production of recombinant proteins. In this report, recombinant beta-glucuronidase (rGUS) was used as a model protein to study the feasibility of using polyethyleneimine (PEI) precipitation to fractionate acidic recombinant proteins from transgenic tobacco. Results showed that rGUS was preferentially precipitated when the PEI dosage was beyond 200mg PEI/g total protein. At 700-800 mg PEI/g total protein, nearly 100% rGUS was precipitated with less than 40% native tobacco proteins co-precipitated. Approximately 85-90% of the rGUS activity could be recovered from the precipitation pellet for an enrichment ratio of 2.7-3.4. As a comparison, anion exchange chromatography (AEX) yielded similar results to PEI precipitation with 66-90% rGUS activity recovered and an enrichment ratio of 1.8-3.1. The rGUS was further purified by an additional hydrophobic interaction chromatographic (HIC) step after precipitation or AEX. Similar results in terms of rGUS activity recovered and enrichment were obtained. The major interfering protein at the end of all purification steps is most likely the Fraction 1 protein ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). The presence of this protein is likely the cause for the varying and somewhat low enrichment ratios, but it may be later removed via a size-exclusion chromatography step. PEI precipitation offers the advantage of allowing significant sample concentration prior to subsequent purification techniques such as chromatography and is much less expensive than chromatographic methods as well. Through direct comparison, this study shows that PEI may be used as an initial fractionation step in replacement of AEX to facilitate the purification of acidic recombinant proteins from transgenic tobacco.     

Solvent-free MALDI-MS for the analysis of biological samples via a mini-ball mill approach

An efficient, low sample load mini-ball mill (MBM) sample preparation procedure was developed for solvent-free MALDI analysis of peptides and proteins. Picomole sample amounts can be handled conveniently, with 30 s grinding times being sufficient. Matrix purity and molar analyte/matrix ratios are not as critical as with methods employing solvent. Ammonium salt is employed for protonation of the peptide and suppression of sodiation. This strategy allows for peptide mapping and other biochemical manipulations to be performed prior to MBM sample preparation and mass analysis. The analysis of bovine serum albumin (66 kDa) yielded good results, indicating that higher molecular weight proteins are accessible. A semi-solvent-free strategy by the MBM sample preparation method is also described.     

Cloning,Expression and Inhibitory Effects on Lewis Lung Carcinoma Cells of rAj-Tspin from Sea Cucumber (Apostichopus japonicus)

In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber (Apostichopus japonicus), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it’s effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.     

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.     

Profile and relative concentrations of fatty acids in corn and soybean seeds from transgenic and isogenic crops

In this work 44 fatty acids, which were analyzed as methyl esters by GC/MS in scan mode, have been determined in genetically modified corn and soybean seeds. Their relative concentrations have been compared with those of isogenic lines grown in the same conditions. Studied compounds comprised saturated and unsaturated fatty acids, including cis/trans isomers and minor fatty acids. A classical soxhlet extraction and an accelerated solvent extraction have been assayed to extract the fatty compounds from seeds and the GC separation has been carried out on a biscyanopropylpolysiloxane chromatographic column. Soxhlet extraction was selected as the most convenient and applied to compare the samples. Specific compounds, which could denote the origin of the crop have not been observed, but for some sample pairs, significant differences have been found in relation to the percentage of certain acids; the highest differences for major acids were 4.1% in corn and 4.8% in soybean. The concentrations of long chain acids such as 24:0, 26:0 and 28:0 were higher in some isogenic lines whereas the concentrations of short chain acids such as 6:0, 8:0, 9:0, 10:0 and 12:0 were higher in their transgenic counterparts.     

Efficient separation of homologous α-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography

Transgenic bovine milk could be a rich source of recombinant human proteins. However, the co-presence of bovine and human homologous proteins can be a challenge for product purification. In this study, the average surface hydrophobicity and electric potential of human α-lactalbumin (HLA) and bovine α-lactalbumin (BLA) were analyzed and compared through the exposure area calculation of different amino acids. Based on the analysis, calcium independent hydrophobic interaction chromatography was selected for separation of recombinant human α-lactalbumin (rHLA) from BLA in transgenic bovine milk. The operating conditions for the best separation of two proteins were predicted by fluorescence data. Three commercially available HIC resins (Butyl Sepharose 4 FF, Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF) were compared. The transgenic milk was skimmed and treated by pH adjustment to remove a large quantity of casein protein. The supernatant was loaded on the hydrophobic interaction chromatographic matrix. The correct elution fraction was further treated with gel filtration chromatography. The overall recovery of rHLA was up to 67.1% with the purity greater than 95%. Circular dichroism spectroscopy (CD) and mass spectrogram (MS) confirmed the native state and glycosylated form of the purified rHLA.     

Analysis of bovine whey proteins in soybean dairy-like products by capillary electrophoresis

The simultaneous separation of bovine whey proteins [alpha-lactalbumin and beta-lactoglobulin (A+B)] and soybean proteins was performed, for the first time, by capillary electrophoresis. Different experimental conditions were tested. The most suitable consisted of 0.050 M phosphate buffer (pH 8) with 1 M urea and 1.2 mg/ml methylhydroxyethylcellulose, UV detection at 280 nm, 15 kV applied voltage, and 30 degrees C temperature. Quantitation of bovine whey proteins in a commercial powdered soybean milk manufactured by adding bovine whey to its formulation was performed using the calibration method of the external standard. Direct injection of a solution of the powdered soybean milk only enabled quantitation of alpha-lactalbumin in the commercial sample. Detection of beta-lactoglobulin (A+B) required acid precipitation of the solution of the sample in order to concentrate bovine whey proteins in the supernatant prior to the analysis of this protein in the whey obtained. Since alpha-lactalbumin could also be quantitated from the injection of the whey, the simultaneous determination of alpha-lactalbumin and beta-lactoglobulin (A+B) was possible upon acid precipitation of the powdered soybean milk solution. Detection limits obtained were 14 microg/g sol. for alpha-lactalbumin and 52 microg/g sol. for beta-lactoglobulin (A+B) which represent protein concentrations about 60 microg/100 g sample for alpha-lactalbumin and 100 microg/100 g sample for beta-lactoglobulin (A+B).     

Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation

The development of novel proteomic technologies that will enable the discovery of disease specific biomarkers is essential in the clinical setting to facilitate early diagnosis and increase survivability rates. We are reporting a shotgun two-dimensional (2D) strong cationic exchange/reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (SCX/RPLC/ESI-MS/MS) protocol for the analysis of proteomic constituents in cancerous cells. The MCF7 breast cancer cell line was chosen as a model system. A series of optimization steps were performed to improve the LC/MS experimental setup, sample preparation, data acquisition and database search protocols, and a data filtering strategy was developed to enable confident identification of a large number of proteins and potential biomarkers. This research has resulted in the identification of >2000 proteins using multiple filtering and p-value sorting. Approximately 1600-1900 proteins had p < 0.001, and, of these, approximately 60% were matched by >or=2 unique peptides. Alternatively, >99% of the proteins identified by >or=2 unique peptides had p < 0.001. When searching the data against a reversed database of proteins, the rate of false positive identifications was 0.1% at the peptide level and 0.4% at the protein level. The typical reproducibility in detecting overlapping proteins across replicate runs exceeded 90% for proteins matched by >or=2 unique peptides. According to their biological function, approximately 200 proteins were involved in cancer-relevant cellular processes, and over 25 proteins were previously described in the literature as putative cancer biomarkers, as they were found to be differentially expressed between normal and cancerous cell states. Among these, biomarkers such PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to MS analysis approximately 42 microg of sample, analyzing 16 SCX peptide fractions, and interpreting approximately 55,000 MS2 spectra. Total MS time required for analysis was 40 h.     

Protein Extraction From Cowpea Tissues for 2-D Gel Electrophoresis and MS Analysis

A method of extraction of proteins from cowpea for two-dimensional electrophoresis is presented. This method avoids loss of protein in the course of sample preparation and results in a fraction that yields reproducible 2-DE protein patterns. The efficiency of this method was demonstrated by comparison of the patterns of protein deposition in developing and mature cowpea seeds. It is also demonstrated that without further processing of the gel piece the proteins present in the spots can be directly digested with trypsin and the peptides subjected to mass spectrometry analysis for identification. Revised: 14 July and 1 August 2005     

Analytical approaches for arsenic determination in air: A critical review

This review describes the different steps involved in the determination of arsenic in air, considering the particulate matter (PM) and the gaseous phase. The review focuses on sampling, sample preparation and instrumental analytical techniques for both total arsenic determination and speciation analysis. The origin, concentration and legislation concerning arsenic in ambient air are also considered. The review intends to describe the procedures for sample collection of total suspended particles (TSP) or particles with a certain diameter expressed in microns (e.g. PM10 and PM2.5), or the collection of the gaseous phase containing gaseous arsenic species. Sample digestion of the collecting media for PM is described, indicating proposed and established procedures that use acids or mixtures of acids aided with different heating procedures. The detection techniques are summarized and compared (ICP-MS, ICP-OES and ET-AAS), as well those techniques capable of direct analysis of the solid sample (PIXE, INAA and XRF). The studies about speciation in PM are also discussed, considering the initial works that employed a cold trap in combination with atomic spectroscopy detectors, or the more recent studies based on chromatography (GC or HPLC) combined with atomic or mass detectors (AFS, ICP-MS and MS). Further trends and challenges about determination of As in air are also addressed.     

Biopharming to increase bioactive peptides in rice seed

The production of high-value pharmaceutical proteins and peptides in transgenic plants is an attractive and economically feasible alternative to conventional mammalian cell, yeast, and bacterial systems. In contrast to vegetative tissues, rice seeds allow higher accumulation of recombinant proteins and long-term stable storage. Rice is not only consumed as a staple food by a majority of the world's population, but is also used as a model monocot for biotechnological manipulation. Daily oral consumption of transgenic rice seeds that accumulate high concentrations of food-derived or synthetic bioactive peptides can be expected to provide a safe, reliable, and consistent oral delivery system that would contribute to the promotion of human health care.     

VIRUS RESISTANCE OF TRANSGENIC TOBACCO PLANTS EXPRESSING TOBACCO MOSAIC VIRUS COAT PROTEIN GENE

An intermediate expressing vector carrying the tobacco mosaic virus (TMV, Chinese common strain) coat protein (CP) gene was constructed by recombinant DNA techniques. The TMV-CP gene was transferred into the tobacco genome via Ti plasmid and a large number of regenerated plants, including both systemic and local lesion hosts for TMV, were obtained. Southern blot analysis revealed that 1-5 copies of the CP gene were integrated into the tobacco genome. RNA and protein analysis demonstrated that the TMV-CP gene was correctly expressed in the transgenic plants. The abundance of TMV-CP mRNA in total leaf RNA accounted for 0.005-0.01%, while the amount of coat proteins reached 0.05-0.2% of the total leaf soluble proteins. Virus challenge experiments showed that the symptom development of virus infection was markedly delayed and the replication as well as the spread of the virus was significantly inhibited in the transgenic plants expressing the TMV-CP gene. Three of these plants were completely protected afte