An efficient extraction method to enhance analysis of low abundant proteins from soybean seed

Large amounts of the major storage proteins, β-conglycinin and glycinin, in soybean (Glycine max) seeds hinder the isolation and characterization of less abundant seed proteins. We investigated whether isopropanol extraction could facilitate resolution of the low abundant proteins, different from the main storage protein fractions, in one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 1D-PAGE of proteins extracted by different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70% and 80%) of isopropanol showed that greater than 30% isopropanol was suitable for preferential enrichment of low abundant proteins. Analysis of 2D-PAGE showed that proteins which were less abundant or absent by the conventional extraction procedure were clearly seen in the 40% isopropanol extracts. Increasing isopropanol concentration above 40% resulted in a decrease in the number of less abundant protein spots. We have identified a total of 107 protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrophotometry (MALDI-TOF-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Our results suggest that extraction of soybean seed powder with 40% isopropanol enriches lower abundance proteins and is a suitable method for 2D-PAGE separation and identification. This methodology could potentially allow the extraction and characterization of low abundant proteins of other legume seeds containing highly abundant storage proteins.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.     

Proteomic Analysis of Allergen and Antinutritional Proteins in Wild and Cultivated Soybean Seeds

In this study, profiles of allergen and antinutritional proteins both in wild (Glycine soja) and cultivated (Glycine max) soybean seeds were compared. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the separation of proteins at two different pH ranges and applied a combined matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) analysis for the identification of proteins. Although overall distribution patterns of the allergen (Gly m Bd 60K, Gly m Bd 30K, Gly m Bd 28K) and antinutritional proteins (trypsin inhibitors and lectin) appeared similar, there was remarkable variation in the number and intensity of the protein spots between wild and cultivated genotypes. The wild genotype showed fifteen polypeptides of Gly m Bd 60K and three polypeptides of trypsin inhibitors. The cultivated genotypes showed twelve polypeptides of Gly m Bd 60K and two polypeptides of trypsin inhibitors. In contrast, the cultivated genotype showed two polypeptides of Gly m Bd 30K and three polypeptides of lectin and the wild genotype showed two and one polypeptides of Gly m Bd 30K and lectin, respectively. Two polypeptides of Gly m Bd 28K were observed in both genotypes. This is the first study reporting the comparative analysis of allergen and antinutritional proteins in both wild and cultivated soybean genotypes using combined proteomic tools.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

IPG-strips versus off-gel fractionation: advantages and limits of two-dimensional PAGE in separation of microsomal fractions of frequently used plant species and tissues

The crucial cellular role of membrane proteins is generally known for all life forms. Depending on the species, tissue, compartment, function and physiological condition, membranes differ in their protein and lipid profiles. Additionally, occurrence of microdomains hampers quantitative protein solubilisation and therefore membrane proteomics remain a major challenge. In the present study sample preparation (TCA/acetone and methanol/chloroform precipitation with and without SDS pre-solubilisation) for two-dimensional PAGE were compared for microsomal fractions of leaves (Arabidopsis thaliana, Nicotiana tabaccum, Pisum sativum) and roots (P. sativum, Zea mays). Generally, pre-solubilisation with SDS impaired the resolution of the gels. All samples showed higher spot yields with TCA/acetone precipitation. Finally, we compared the results of conventional 2D-PAGE (IPG/SDS-PAGE) and the combination of off-gel fractionation in the first-dimension, 10% urea-SDS-PAGE in the second-dimension. Results showed that more spots are present in the alkaline pH range after off-gel fractionation then on conventional 2D-PAGE. For the first time, off-gel fractionation was combined with SDS/SDS-PAGE and BAC/SDS-PAGE to improve the resolution after off-gel fractionation. Transmembrane domains and GRAVY were calculated for all significantly identified spots resulting from the MALDI-TOF-TOF mass spectrometry showing that in the second dimension after off-gel fractionation 10.3% more transmembrane proteins were identified compared to IPG/SDS-PAGE.     

柑橘叶片和果皮总蛋白质提取及双向电泳体系的建立

以春甜橘(Citrus reticulata Blanco‘Chuntianju’)果皮和叶片为材料,分别用Tris-HCl、尿素/硫脲(Thi/Urea)、三氯乙酸/丙酮(TCA)和酚(Phe)等4种方法提取柑橘总蛋白质,从蛋白质产量、单向SDS-PAGE和双向电泳等方面进行比较。结果表明,4种方法的分离效果存在较大差异,不论是以柑橘叶片还是果皮为材料,均以TCA法最好,且双向电泳图谱分辨率较好,蛋白点清晰、均匀、基本没有条纹,且蛋白点多。这说明TCA法不仅能很好地去除柑橘果皮、叶片中存在的大量干扰物质,而且还能得到稳定的蛋白点。     

Determination of Seed Storage Proteins and Total Isoflavones in Wild and Cultivated Soybeans

Two soybean components namely, storage proteins and isoflavone content in a wild and three cultivated soybean genotypes were characterized and compared. The storage proteins, β-conglycinin and glycinin were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and two major storage proteins and their subunits were characterized using mass spectrometry. The three isoflavones, aglycon and the nine conjugated forms were separated by HPLC (high performance liquid chromatography) and identified by comparison of retention time, ultraviolet and mass spectral analyses. Comparison between the number of 2D-PAGE protein spots of the storage protein subunits and HPLC area of twelve isoflavones was also evaluated. The analysis of proteins and isoflavones from the wild genotype and the three cultivated genotypes suggested possible interactions between proteins and isoflavones. The same wild genotype, which showed significant statistical differences in β-conglycinin and glycinin protein profiles also revealed considerable reduction in total isoflavones (> 55%) content.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

Investigations into charge heterogeneity of wool intermediate filament proteins

Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

非洲山毛豆叶片蛋白组双向电泳样品制备方法的建立

以非洲山毛豆叶片为材料,对非洲山毛豆总蛋白质3种提取方法(TCA/丙酮沉淀法、尿素/硫脲法和酚-甲醇/醋酸铵沉淀法)以及3种蛋白裂解液进行比较分析。结果表明,采用酚-甲醇/醋酸铵沉淀法提取非洲山毛豆叶片总蛋白,用蛋白裂解液(7mol/L尿素,2mol/L硫脲,4%CHAPS,40mmol/LTris-base,1%Bio-LytepH3.5-10,65mmol/LDTT)裂解蛋白1h,2-DE图谱分离到的蛋白点效果最好。此方法适合于色素、多酚及黄酮类次生代谢物含量较多的非洲山毛豆叶片总蛋白制备方法。     

衫木叶片蛋白质组的双向电泳技术优化

为建立适用于杉木(Cunninghaimia lanceolata)叶片蛋白质组研究的双向电泳技术,对杉木叶片蛋白质的溶解方法、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化。结果表明,杉木叶片蛋白质主要分布在pH4-7范围;裂解液中含有硫脲(2mmol/L)才能较充分地溶解蛋白,DTT浓度为60mmol/L、上样量1.5mg时得到的图谱分辨率较好且蛋白斑点分布均匀、清晰,拖尾现象明显减少,平衡液Ⅱ中碘代乙酰胺浓度为450mg(15ml)-1时能提高图谱分辨率;采用与质谱兼容的考马斯亮兰进行染色,得到近700个蛋白点。     

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.     

Initial proteome analysis of mature barley seeds and malt

Several barley (Hordeum vulgare) cultivars are used in the production of malt for brewing. The malt quality depends on the cultivar, its growth and storage conditions, and the industrial process. To enhance studies on malt quality, we embarked on a proteome analysis approach for barley seeds and malt. The proteome analysis includes two-dimensional (2-D) gel electrophoresis, mass spectrometry, and bioinformatics for identification of selected proteins. This project initially focused on proteins in major spots in the neutral isoelectric point range (pI 4-7) including selected spots that differ between four barley cultivars. The excellent malting barley cultivar Barke was used as reference. Cultivar differences in the 2-D gel spot patterns are observed both at the seed and the malt level. In seed extracts one of the proteins causing variations has been identified as an alpha-amylase/trypsin inhibitor. In malt extracts multiple forms of the alpha-amylase isozyme 2 have been identified in varying cultivar characteristic spot patterns. The present identification of proteins in major spots from 2-D gels includes 27 different proteins from 42 spots from mature seed extract, while only three specific proteins were identified by analysing 13 different spots from the corresponding malt extract. It is suggested that post-translational processing causes the same protein to occur in different spots.     

Acoustic technology for high-performance disruption and extraction of plant proteins

Acoustic technology shows the capability of protein pellet homogenization from different tissue samples of soybean and rice in a manner comparable to the ordinary mortar/pestle method and far better than the vortex/ultrasonic method with respect to the resolution of the protein pattern through two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). With acoustic technology, noncontact tissue disruption and protein pellet homogenization can be carried out in a computer-controlled manner, which ultimately increases the efficiency of the process for a large number of samples. A lysis buffer termed the T-buffer containing TBP, thiourea, and CHAPS yields an excellent result for the 2D-PAGE separation of soybean plasma membrane proteins followed by the 2D-PAGE separation of crude protein of soybean and rice tissues. For this technology, the T-buffer is preferred because protein quantification is possible by eliminating the interfering compound 2-mercaptoethanol and because of the high reproducibility of 2D-PAGE separation.