Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.

     

温敏核不育水稻花药蛋白质组初步分析

采用固相pH梯度 SDS聚丙烯酰胺双向凝胶电泳对温敏核不育水稻 96 4 2S可育与不育条件下减数分裂期花药总蛋白进行了分离 ,通过银染显色 ,获得了分辨率和重复性较好的双向电泳图谱 .PDQuest 2DE图像分析软件可识别约 10 0 0个蛋白质点 .蛋白质点在 2D胶上的重复性为 :沿等电聚焦方向偏差为 1 4 5± 0 2 3mm(n =8) ,沿SDS PAGE方向偏差为 :1 15± 0 17mm(n =8) .对两种育性不同样品的 2D胶上部分共有的蛋白质点 ,采用基质辅助激光解析电离飞行时间质谱 (matrixassistedlaserdesorption ionizationtimeofflightmassspctrometry ,MALDI TOF MS)进行了肽质谱指纹图分析 .通过采用PeptIdent软件对SWISS PROT数据库的查询 ,有 5 0个蛋白质点在数据库得到归属鉴定 .对育性不同的2种样品 2D较上明显差异的蛋白质点进行了分析鉴定 .在不育变化为可育的过程中 ,明显表达上调的蛋白质点包括几丁质酶 ,酸性磷酸酶 ,胞浆激酶 ,谷蛋白前体 ,以及ESTSC72 61蛋白 ,明显下调的蛋白质包括β expansin前体 ,谷氨酸氨甲酰转移酶和 1种未知功能的蛋白质     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour,one of the most popular aeroallergens in China,were analyzed by two-dimensional electrophoresis in the current study.The proteins were extracted by Trichloracetic acid (TCA) method,and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension.The spots of proteins were visualized by staining with Coomassie Brilliant Blue.After analysis with software (ImageMaster 2D),122 different proteins were detected;isoelectric point (pI),Molecular weight (MW) and relativevolume of each protein in the pollen were also discovered.This is the first high-resolution,two-dimensional protein map of the pollen ofHumulus scandens Lour in China.Our finding has built a solid foundation for identification,characterization,gene cloning and standardization of allergenic proteins in the pollen ofHumulus scandens Lour for further studies.     

中国武汉地区葎草花粉变应原蛋白质组分的双向电泳分析

应用蛋白质双向电泳的分析技术对武汉地区生长的主要过敏原———葎草(Humulus scandensLour.)花粉的蛋白质组分进行了分析。应用三氯乙酸法提取葎草花粉总蛋白质,通过等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳分析获得了完整的葎草花粉全蛋白质图谱。应用专业分析软件(Im ageM aster 2D)对电泳图谱分析表明:通过等电聚焦和第二向SDS-PAGE电泳,有122个不同的蛋白质组分被检测出来,并进一步确定每个蛋白质相应的分子量、等电点和相对含量。研究中获得的高分辨率的双向电泳图谱是我国葎草花粉变应原蛋白质第一张完整的蛋白质图谱,将为今后葎草花粉中致敏蛋白质的检测、分离、基因克隆和变应原的标准化奠定基础。     

A proteomic approach for investigation of photosynthetic apparatus in plants

The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.     

湖南烙铁头蛇毒蛋白组分的双向电泳分析

烙铁头蛇是世界上剧毒的蛇种之一,其所携带的毒素能够导致严重的机体损伤。应用蛋白质双向电泳技术,对湖南烙铁头蛇蛇毒蛋白的蛋白质组分进行分析。通过等电聚焦和SDS-PAGE凝胶电泳分析获得完整的烙铁头蛇毒全蛋白质的图谱,经胶体考马斯亮蓝染色后,应用PDQuest软件对蛋白表达谱进行分析。通过等电聚焦和SDS-PAGE凝胶电泳有83个蛋白质组分被检测出来。其中大约90.00%的蛋白质的相对分子质量(Mr)分布在15～45 kDa之间,大约72.29%的蛋白质等电点(pI)在4.0～7.0之间。通过对烙铁头蛇毒的蛋白组学研究,获得其蛇毒蛋白质组分的表征特点,为后续进一步研究各组分的身份和潜在功能奠定基础,既可以提出新的治疗方案又可以为新的药理应用提供宝贵资源。     

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.     

Proteomic tools to characterize the protein fraction of Equidae milk

The principal components of the protein fraction in pony mare's milk have been successfully identified and partially characterized using proteomic tools. Skimmed pony mare's milk was fractionated by either reversed phase-high-performance liquid chromatography (RP-HPLC) on a C4 column or a bi-dimensional separation technique coupling RP-HPLC in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) in the second dimension (two-dimensional RP-HPLC/SDS-PAGE). The fractions thus obtained were analyzed by Edman N-terminal microsequencing and mass determination, with or without tryptic digestion, on a matrix-assisted laser desorption/ionization-time of flight spectrometer. Based on the sequence and molecular mass information obtained, identifications were achieved through a protein database search using homology or pattern research algorithms. This methodological approach was shown to be rapid, efficient and reliable in identifying the principal proteins in pony mare's milk. kappa-, alpha(s1)-, alpha(s2)-, and beta-casein, lysozyme C, alpha-lactalbumin and beta-lactoglobulin I and II were thus identified. alpha(s1) and beta-caseins displayed polymorphic patterns, probably due to alternative splicing processes leading to casual exon skipping events involving exons 7 and 14 in alpha(s1)-casein and exon 5 in beta-casein. Edman N-terminal microsequencing over 35 amino acid residues, for pony alpha(s1)-casein, clearly demonstrated the occurrence, in Equidae, of a splicing pattern similar to that reported in rodents, characterized by the constitutive outsplicing of exon 5. Pony mare's milk SDS-PAGE and RP-HPLC patterns were compared with those obtained for other milks (cow, goat and human), as were the relative levels of caseins and major whey proteins in these milks. Our results provide further evidence to support the notion that Equidae milk is closer to human breast milk than milk from bovine and caprine with respect to the casein and lysozyme C contents and casein/whey proteins ratio.     

Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics

Despite the importance of membranes in any living system, the global analysis of membrane subproteomes is still a common obstacle. In particular, the widely used 2-DE technique consisting of IEF in the first dimension and SDS-PAGE in the second dimension has some major drawbacks regarding the separation of hydrophobic proteins. Therefore, we applied an alternative electrophoretic technique for separating membrane proteins: two-dimensional BAC/SDS electrophoresis (2-DB) using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride in the first and the anionic detergent SDS in the second dimension. The use of 2-DB resulted in an improved separation of hydrophobic proteins. Thus, extremely hydrophobic proteins such as cytochrome-c oxidase subunit I with a grand average hydrophobicity (GRAVY) index of 0.74 and a total of 12 known transmembrane domains (TMD) or Sec61alpha with a GRAVY index of 0.56 and a total of ten known TMD could be identified by MS/MS analyses of protein spots derived from 2-DB gels.     

Technical improvement to 2D-PAGE of rice organelle membrane proteins

Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.     

Soluble pathogenesis-related proteins ofCapsicum annuum leaves induced by cucumber mosaic virus infection

Eight major pathogenesis related (PR) proteins were found in a soluble extract of cucumber mosaic virus (CMV) infectedCapsicum annuum leaves. None of them was present in the soluble extract of sham-inoculated controls. The proteins were detected by two-dimensional polyacrylamide gel electrophoresis using (2D-PAGE)PhastSystem, native-PAGE in the first dimension and SDS-PAGE in the second. Two major acidic PR-proteins were identified on the basis of their relative molecular mass (M_r), PR1 of 15.3 kD and PR2 of 29 kD. Six proteins were basic and were identified as PRla of 15.9 kD, PRlb of 15.0 kD, PRlc of 15.9 kD, PR2 of 27.0 kD, PR3 of 36.3 kD and PR4 of 48.8 kD.     

杀雄剂SQ-1诱导小麦雄性不育花粉粒差异蛋白质组学研究

采用固相pH梯度/SDS-PAGE双向凝胶电泳对经杀雄剂SQ-1处理和未处理的小麦(Triticum aestivum)成熟期花粉总蛋白质进行了分离, 银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 通过PDQuest 2DE图像软件的分析, 在等电点4～7之间可识别350个以上较为清晰的蛋白质点, 其中差异表达明显的蛋白质点数为21个. 将11个差异点采用基质辅助激光解析电离飞行时间质谱进行了肽质量指纹图谱分析, 采用Mascot软件在Swiss-prot数据库查询, 鉴定出了7个蛋白质, 它们分别是液泡转化酶、动力蛋白轻链TCTEX-1、锰超氧化物歧化酶、果糖-1,6-二磷酸醛缩酶、抗坏血酸过氧化物酶、凝集素蛋白激酶和一种未知功能的蛋白. 对已知蛋白的功能进行分析, 推测杀雄剂SQ-1诱导小麦雄性不育可能与能量代谢失衡、淀粉合成受抑制、活性氧积累、细胞凋亡以及花器官发育调节基因作用失控等有关.     

Analysis of normal and neoplastic lymphocyte surface-labeled proteins by two-dimensional polyacrylamide gel electrophoresis

Normal and neoplastic murine and human lymphocytes were surface-labeled by lacto-peroxidase-catalyzed radioiodination, and the cell lysates were subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analyses, combining isoelectric focusing in the first dimension and sodium dodecyl sulfate-PAGE (SDS-PAGE) in the second dimension. 2D-PAGE autoradiogram patterns were reproducible and reflected differences in cell types. A string of spots with a Mr of 100K was tentatively identified as a new T-cell marker (Tp100) which was present in all murine and human T cells examined including human T lymphomas. Murine and human B cells displayed markers characteristic to B cells of each species with some similarities between them. Human lymphomas and murine cell lines showed markers which were absent or only weakly visible in normal cells. Thus, 2D-PAGE analysis of lymphocyte surface proteins proved to be a method useful for searching for various markers.     

Analysis of total proteins in pollen of Humulus scandens Lour in Wuhan Region of China by two-dimensional electrophoresis

Total proteins in the pollen of Humulus scandens Lour, one of the most popular aeroallergens in China, were analyzed by two-dimensional electrophoresis in the current study. The proteins were extracted by Trichloracetic acid (TCA) method, and then separated by isoelectric focusing as the first dimension and SDS-PAGE as the second dimension. The spots of proteins were visualized by staining with Coomassie Brilliant Blue. After analysis with software (ImageMaster 2D), 122 different proteins were detected; isoelectric point (pI), Molecular weight (MW) and relative volume of each protein in the pollen were also discovered. This is the first high-resolution, two-dimensional protein map of the pollen of Humulus scandens Lour in China. Our finding has built a solid foundation for identification, characterization, gene cloning and standardization of allergenic proteins in the pollen of Humulus scandens Lour for further studies. Translated from Journal of Wuhan Botanical Research, 2006, 24(1): 58–62 [译自: 武汉植物研究]     

The synaptic vesicle proteome: a comparative study in membrane protein identification

A proteomic analysis of the synaptic vesicle was undertaken to obtain a better understanding of vesicle regulation. Synaptic vesicles primarily consist of integral membrane proteins that are not well resolved on traditional isoelectric focusing/two-dimensional gel electrophoresis (IEF/2-DE) gels and are resistant to in-gel digestion with trypsin thereby reducing the number of peptides available for mass spectrometric analysis. To address these limitations, two complementary 2-DE methods were investigated in the proteome analysis: (a) IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) for resolution of soluble proteins and, (b) Benzyl hexadecyl ammonium chloride/SDS-PAGE (16-BAC/SDS-PAGE) for resolution of integral membrane proteins. The IEF/SDS-PAGE method provided superior resolution of soluble proteins, but could only resolve membrane proteins with a single transmembrane domain. The 16-BAC/SDS-PAGE method improved separation, resolution and identification of integral membrane proteins with up to 12 transmembrane domains. Trypsin digestion of the integral membrane proteins was poor and fewer peptides were identified from these proteins. Analysis of both the peptide mass fingerprint and the tandem mass spectra using electrospray ionization quadrupole-time of flight mass spectrometry led to the positive identification of integral membrane proteins. Using both 2-DE separation methods, a total of 36 proteins were identified including seven integral membrane proteins, 17 vesicle regulatory proteins and four proteins whose function in vesicles is not yet known.     

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.     

Tricine-SDS-PAGE

Tricine-SDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1-2 d.     

Mass spectrometry identification of NASP binding partners in HeLa cells

Nuclear autoantigenic sperm protein (NASP) is a linker histone binding protein that is cell-cycle regulated. Synchronized HeLa cells are delayed in progression through the G1/S border when transiently transfected to overexpress full-length NASP, but not the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS). The purpose of the current study was to identify possible NASP-associated proteins in HeLa cell nuclei that could elucidate NASP's influence on the cell cycle and chromatin remodeling. For this purpose, we employed a new approach: mass spectrometry identification of initially cross-linked proteins after their separation in a second dimension by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Of the twelve proteins identified, three appear to be relevant to NASP's function: heat shock protein 90 (HSP90), DNA-activated protein kinase, and ATP-dependent DNA helicase II (70-kDa subunit). Individual protein-protein interactions were tested by immunoprecipitation techniques. This new method can be used for expedited identification of binding partners of different proteins in enriched fractions and as a complementary or alternative strategy to the yeast two-hybrid system and immunoprecipitation methods.