Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources. The proteome of Escherichia coli D21 grown to either mid-exponential or stationary phase, and the membrane proteome from established breast cancer cell lines BT474 and MCF7 were used as model systems in these experiments. In both experiments, the numbers of total identified proteins are at least twice the numbers identified from a single tagging cycle. The sample complexity can be effectively reduced with corresponding increases in protein identification using the multistep method. The strategy described here represents a potentially powerful technique for large-scale qualitative and quantitative proteome research.     

Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF

A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.     

Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.     

Evaluation of Protein Extraction Methods for Vitis vinifera Leaf and Root Proteome Analysis by Two-Dimensional Electrophoresis

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.     

苹果属植物树皮组织总蛋白提取及分离方法的建立

为了建立适于苹果属植物树皮组织总蛋白提取的技术方法, 以8年生华月苹果(Malus domestica)树枝条为试材, 通过比较不同提取方法(TCA-丙酮沉淀法(A)、甲醇/醋酸铵沉淀法(B)和改良的Tris-酚抽提方法(C))并优化提取条件, 确立了最适提取及分离方法为改良的Tris-酚抽提方法。在2-DE分离时, 该方法所获得的样品图谱中蛋白点总数为993个, 明显多于TCA-丙酮沉淀(418个)和甲醇醋酸铵沉淀(674个)方法, 并且与其它两种方法相比, 该方法获得的图谱背景更清晰, 蛋白点聚焦效果更好。此外, 经过3个梯度上样量的图谱分离效果比较, 确定了800 μg为本研究中2-DE分析的理想上样量。另外, 为了验证该提取及分离方法的可行性, 进一步对蛋白质表达谱中的部分蛋白点进行了质谱分析, 且这些蛋白点均得到了成功鉴定。该研究通过优化总蛋白提取方法及样品上样量等条件, 获得了理想的双向电泳分离图谱, 为苹果属植物树皮组织材料的蛋白质组学研究奠定了基础。     

Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE

The number of proteomics studies concerning human brain samples has been increasing in recent years, in particular in the discovery of biomarkers for neurological diseases. The human brain samples are obtained from brain banks, which are interested in providing high quality human nervous tissue. In order to provide brain banks as well as scientists working in the proteomics field with measures for tissue quality, the critical factors after death, the effect of post-mortem interval (PMI) and storage temperature on the human brain proteome were investigated. This study was focused on the gray matter of the frontal cortex. The PMI was artificially prolonged from the time of autopsy (2 h after death) by storing samples at 4 degrees C or room temperature over 18, 24, and 48 h. The samples were analyzed by 2-D DIGE using a pH 4-7 gradient, revealing a time course of quantitative protein changes. The degradation of three proteins, peroxiredoxin-1, stathmin, and glial fibrillary acidic protein were further confirmed by Western-blot analysis. Proteins vulnerable to PMI were analyzed by the 2-D DIGE analysis of cortex samples from three donors, and were derived from a variety of functional groups, including metabolic, structural, stress response, antioxidants, synaptosomal, and neuronal proteins.     

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.     

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.     

植物蛋白质组学研究若干重要进展

植物蛋白质组学近年来正从定性向精确定量蛋白质组学的方向发展。国际上近两年发表的约160篇研究论文报道了利用不断改进的双向电泳结合生物质谱技术、多维蛋白质鉴定技术,以及包括双向荧光差异凝胶电泳、幅N体内代谢标记、同位素标记的亲和标签、同位素标记相对和绝对定量等在内的第2代蛋白质组学技术,对植物组织（器官）与细胞器、植物发育过程和植物响应环境胁迫的蛋白质组特征,以及植物蛋白质翻译后修饰和蛋白质相互作用等方面的研究成果。该文对上述报道进行总结,综述了2007年以来植物蛋白质组学若干重要问题研究的新进展。     

植物蛋白质组学研究若干重要进展

植物蛋白质组学近年来正从定性向精确定量蛋白质组学的方向发展。国际上近两年发表的约160篇研究论文报道了利用不断改进的双向电泳结合生物质谱技术、多维蛋白质鉴定技术, 以及包括双向荧光差异凝胶电泳、15N体内代谢标记、同位素标记的亲和标签、同位素标记相对和绝对定量等在内的第2代蛋白质组学技术, 对植物组织(器官)与细胞器、植物发育过程和植物响应环境胁迫的蛋白质组特征, 以及植物蛋白质翻译后修饰和蛋白质相互作用等方面的研究成果。该文对上述报道进行总结, 综述了2007年以来植物蛋白质组学若干重要问题研究的新进展。     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

Comparison of Protein-Extraction Methods for Gills of the Shore Crab,Carcinus maenas (L.), and Application to 2DE

As it is well-established that protein extraction constitutes a crucial step for two-dimensional electrophoresis (2DE), this work was done as a prerequisite to further the study of alterations in the proteome in gills of the shore crab Carcinus maenas under contrasted environmental conditions. Because of the presence of a chitin layer, shore crab gills have an unusual structure. Consequently, they are considered as a hard tissue and represent a challenge for optimal protein extraction. In this study, we compared three published extraction procedures for subsequent applications to 2DE: the first one uses homogenization process, the second one included an additional TCA-acetone precipitation step, and finally, the third one associated grinding in liquid nitrogen (N₂) and TCA-acetone precipitation. Extracted proteins were then resolved using 1DE and 2DE. Although interesting patterns were obtained using 1DE with the three methods, only the one involving grinding in liquid N₂ and TCA-acetone precipitation led to proper resolution after 2DE, showing a good level of reproducibility at technical (85%) and biological (84%) levels. This last method is therefore proposed for analysis of gill proteomes in the shore crab.     

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.     

Native-DIGE: a new look at the mitochondrial membrane proteome

Respiratory chain proteins play a pivotal role in mitochondrial metabolism and thereby in the aging process. Differential display of the mitochondrial proteome reveals the abundance changes occurring in proteins as response to complex events such as senescence and aging. However, there is an absolute need to implement a detection technique that could potentially encompass the hydrophobic and very basic membrane proteins, along with the soluble ones. It is also important to assess protein-protein interactions, besides changes in abundance. Native-difference gel electrophoresis (DIGE) is an approach that facilitates sensitive quantitative assessment of changes in membrane and soluble proteins. It stretches the boundaries of detecting abundance changes to protein-protein interactions for interpretation of a proteome in a more "meaningful" way. Here we evaluate the benefits of blue-native fluorescence DIGE as a method in differential quantitative proteomics with a focus on critical issues for application and experimental design.     

长双歧杆菌BBMN68 可溶性蛋白质图谱的建立

为了建立长双歧杆菌BBMN68蛋白质图谱,采用双向电泳的方法建立了2-D参考图谱,通过MALDI-TOF/MS质谱鉴定和数据库搜索,鉴定到206个蛋白质(占长双歧杆菌BBMN68基因预测总蛋白的11.4%)。通过2-D胶分析,共有800±15(对数期)和800±20(稳定期)个蛋白质,其中282个蛋白点成功鉴定,代表206个不同的蛋白质。另外,分析了实验鉴定蛋白质的等电点和分子量,蛋白功能,密码子偏好性,蛋白质疏水性以及蛋白质细胞定位的分析。研究结果为长双歧杆菌的比较蛋白质组学研究提供了参考图谱和蛋白质基础信息数据。     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Biomarker discovery from the top down: Protein biomarkers for efficient virus transmission by insects (Homoptera: Aphididae) discovered by coupling genetics and 2-D DIGE

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are vectored by aphids. The identification of vector proteins mediating virus transmission is critical to develop sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Previously, we applied 2-D DIGE to an aphid filial generation 2 population to identify proteins correlated with the transmission phenotype that were stably inherited and expressed in the absence of the virus. In the present study, we examined the expression of the DIGE candidates in previously unstudied, field-collected aphid populations. We hypothesized that the expression of proteins involved in virus transmission could be clinically validated in unrelated, virus transmission-competent, field-collected aphid populations. All putative biomarkers were expressed in the field-collected biotypes, and the expression of nine of these aligned with the virus transmission-competent phenotype. The strong conservation of the expression of the biomarkers in multiple field-collected populations facilitates new and testable hypotheses concerning the genetics and biochemistry of virus transmission. Integration of these biomarkers into current aphid-scouting methodologies will enable rational strategies for vector control aimed at judicious use and development of precision pest control methods that reduce plant virus infection.     

Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma

The plasma proteome has a wide dynamic range of protein concentrations and is dominated by a few highly abundant proteins. Discovery of novel cancer biomarkers using proteomics is particularly challenging because specific biomarkers are expected to be low abundance proteins with normal blood concentrations of low nanograms per milliliter or less. Conventional, one- and two-dimensional proteomic methods including 2D PAGE, 2D DIGE, LC-MS/MS, and LC/LC-MS/MS do not have the capacity to consistently detect many proteins in this range. In contrast, new higher dimensional (Hi-D) separation strategies, utilizing more than two dimensions of fractionation, can profile the low abundance proteome.