Effective protein extraction protocol for proteomics studies of Jerusalem artichoke leaves

Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2DE) analysis in Jerusalem artichoke (Helianthus tuberosus L.), three different protein extraction methods—trichloroacetic acid/acetone, Mg/NP‐40, and phenol/ammonium acetate—were evaluated using Jerusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2DE analysis. Proteins highly abundant in leaves, such as Rubisco, are typically problematic during leaf 2DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than Mg/NP‐40 and phenol/ammonium acetate in Jerusalem artichoke leaf 2DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2DE analysis of Jerusalem artichoke.     

Comparative evaluation of five protocols for protein extraction from stony corals (Scleractinia) for proteomics

Corals especially the reef‐building species are very important to marine ecosystems. Proteomics has been used for researches on coral diseases, bleaching and responses to the environment change. A robust and versatile protein extraction protocol is required for coral proteomics. However, a comparative evaluation of different protein extraction protocols is still not available for proteomic analysis of stony corals. In the present study, five protocols were compared for protein extraction from stony corals. The five protocols were TRIzol, phenol‐based extraction (PBE), trichloroacetic acid (TCA)‐acetone, glass bead‐assisted extraction (GBAE) and a commercially available kit. PBE, TRIzol and the commercial kit were more robust for extracting proteins from stony corals. The protein extraction efficiency and repeatability, two dimensional electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS) were employed to evaluate the protocols. The results indicated that PBE protocol had the better protein extraction efficiency than the others. Protein extraction coverage varied among the procedures. Each protocol favored for certain proteins. Therefore, it is very important for coral proteomic analysis to select a suitable protein protocol upon the experimental design. In general, PBE protocol can be the first choice for extracting proteins from stony corals.     

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

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

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Protein extraction from Phoenix dactylifera L. leaves, a recalcitrant material, for two-dimensional electrophoresis

This work was aimed at optimizing a protein extraction procedure for date palm (Phoenix dactylifera L.) leaves, a highly recalcitrant plant tissue for 2-DE. Five protein extraction protocols based on different protein precipitation agents (TCA/acetone vs. phenol (Ph) methods) and protein resolubilization methods (physical treatments, e.g., sonication, shaking and/or heating) were tested. Ph/SDS extraction with methanol/ammonium acetate precipitation, followed by DOC preincubation and TCA/acetone precipitation and, finally, solubilization by shaking in rehydration solution was found to be the best protein extraction method. We conclude that DOC with TCA/acetone precipitation step eliminates interfering compounds, thus allowing efficient resolubilization of date palm leaf proteins. This method could be appropriate for proteomic studies such as date palm colonization by entomopathogenic fungi.     

Evaluation of sample extraction methods for proteomics analysis of green algae Chlorella vulgaris

Many protein extraction methods have been developed for plant proteome analysis but information is limited on the optimal protein extraction method from algae species. This study evaluated four protein extraction methods, i.e. direct lysis buffer method, TCA‐acetone method, phenol method, and phenol/TCA‐acetone method, using green algae Chlorella vulgaris for proteome analysis. The data presented showed that phenol/TCA‐acetone method was superior to the other three tested methods with regards to shotgun proteomics. Proteins identified using shotgun proteomics were validated using sequential window acquisition of all theoretical fragment‐ion spectra (SWATH) technique. Additionally, SWATH provides protein quantitation information from different methods and protein abundance using different protein extraction methods was evaluated. These results highlight the importance of green algae protein extraction method for subsequent MS analysis and identification.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

Optimization of protein extraction and solubilization for mature grape berry clusters

Protein extraction from grape berries has been challenging, particularly in mature berries, which can have sugar concentrations as high as 26%. Grape skins and seeds contain large amounts of polyphenols, which can also interfere with efficient protein extraction. In plants, two extraction protocols, TCA/acetone-based and phenol-based methods, have been mainly used to extract proteins from different organs or tissues on many species. However, few results have been reported for grape berry clusters. We wanted to determine which of these protocols was optimal for berry clusters in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. Four protocols, derived from either TCA/acetone or phenol procedures, were tested on mature Cabernet Sauvignon whole berry clusters. The phenol-based protocols were superior to the TCA/acetone methods, showing larger protein yields and greater spot resolution on 2-D gels. One method was clearly superior to the rest, a phenol-based extraction method combined with resuspension in the presence of both urea and thiourea as chaotropes. A total of 81 spots were excised and identified following MALDI-TOF/TOF MS analyses. Their identification helped further characterize the specificity of each extraction procedure.     

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.)

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI‐protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.     

Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis

Sample preparation is a fundamental step in proteomic methodologies. The quality of the results from a proteomic experiment is dependent on the nature of the sample and the properties of the proteins. In this study, various pre-treatment methods were compared by proteomic analysis; we analysed various rat brain structures after chloroform/methanol, acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the supernatant was also examined by 2-DE. We found that for four of the rat brain structures, precipitation with chloroform/methanol and acetone delivered the highest protein recovery for top-down proteomic analysis; however, TCA precipitation resulted in good protein separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation also gave high efficiency of protein recovery if prior sonication procedure was performed.     

Characterization of quinoa seed proteome combining different protein precipitation techniques: Improvement of knowledge of nonmodel plant proteomics

A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl₃/double‐distilled H₂O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in‐solution digested and the resulting peptides were analyzed by nano‐liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high‐throughput studies on Chenopodium Quinoa.     

Proteomic platform for the identification of proteins in olive (Olea europaea) pulp

The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

A systematic evaluation of protocols for a proteomics analysis of (lyophilized) fruit tissues

This study represents a systematic evaluation of protocols for protein extraction and cleanup for fruit proteomic analysis. Procedures were optimized using pooled lyophilized banana fruit pulp, which is known to be particularly tricky due to high concentrations of soluble polysaccharides, phenolics, and other substances that interfere with protein extraction and purification. A total of 18 combinations of three protein extraction procedures (SDS‐based, Triton X‐100‐based, and phenol‐based), three protein precipitating agents (ammonium acetate/methanol, TCA/acetone, and acetone), and two resolubilization buffers (classical Rabilloud and the so‐called R2D2) were compared for total protein yields and efficiency of recovery. The results demonstrate that while losses in total recovered protein are unavoidable, the degree of these losses depends on the method combinations used. Combinations based on buffer‐saturated phenol always gave the highest yields, and overall recovery and purity was highest when acetone was combined with the R2D2 buffer for protein purification and concentration. Comparative 2D‐PAGE analysis confirmed that this method combination produced high‐quality and reproducible gels and the largest numbers of spots per gel. The usefulness of this methodology was demonstrated on ripe fruits from several other species and shown to give excellent results.     

Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis

This paper investigates the preparation of Fasciola hepatica samples for two-dimensional electrophoresis (2-DE). Whole samples were prepared by both hot sodium dodecyl sulfate (SDS) solubilisation and precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants and to inactivate endogenous proteases. Sample preparation had a marked influence on the 2-DE gel profile. TCA precipitation resulted in no measurable improvement in the profile observed, compared to the untreated control. Solubilisation of sample with hot SDS increased the number of protein spots, as did TCA precipitation with the addition of phosphotungstic acid. The preparation of excretory-secretory (ES) products poses problems due to both high salt concentrations and low protein concentration. All precipitation methods used to overcome this gave similar profiles, except acetone alone, which caused depletion of the larger proteins. TCA in acetone gave the best result, similar to that obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sample. This problem was alleviated by differential solubilisation, which also resulted in the enrichment of some proteins.     

基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取,酶解肽段脱盐后用离线反相-反相二维液相色谱分离,然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析,共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响,结果表明：在二维液相色谱条件下,酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914,分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下,酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比,酚法不但鉴定到的蛋白质数量多,而且能够鉴定到一些极端蛋白质,如酸性、碱性及高等电点的蛋白质。此外,对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类,发现3种方法鉴定到的蛋白质的功能存在互补性,但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑,同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.     

蛋白质组学技术筛选与鉴定镉诱导下褐云玛瑙螺肝脏的差异蛋白质

比较丙酮/TCA沉淀法和直接裂解法,优化提取与分离褐云玛瑙螺(Achatina fulica, AF)肝脏全蛋白.采用丙酮/TCA沉淀法,可获得约600个蛋白质斑点.用0.5 mg/L CdCl2溶液浸泡后的去梗小白菜喂养AF,并作为镉盐诱导AF肝脏表达应激蛋白质的实验材料.采用蛋白质组学技术筛选出由镉盐诱导AF肝脏表达的14个差异蛋白质斑点,并用肽质量指纹图谱技术(peptide mass fingerprinting, PMF)和数据库比对法初步鉴定出7种差异蛋白质,其中部分为热激蛋白(heat shock protein)、甲基转移酶(Methyltransferase)、三磷酸腺苷结合盒子转运体(ABC transporter)、钼酸盐转运子亚基(molybdate transporter subunit)和磷酸甘油酸变位酶(phosphoglycerate mutase).差异蛋白质均参与镉盐代谢,并适合作为监测土壤或食物中镉污染程度及危害性的指示蛋白质.     

Evaluation of sample preparation protocols for proteomic analysis of sunflower leaves

Six protocols for extraction of proteins from sunflower (Helianthus annuus L.) leaves were evaluated for their abilities in both removing interferents and attaining the best resolution in two-dimensional gel electrophoresis. “Classical” phenol extraction followed by precipitation with ammonium acetate in methanol displayed the most efficient protocol, which allowed the detection of 244 protein spots with ca. 485 μg of protein in gel electrophoresis. Tandem mass spectrometry was performed to identify proteins in 61 spots, and cross species identification was used for this task. Proteins from twenty two spots were identified, and 12 of these proteins are up to now not included into the ExPASy sunflower protein databank.     

An optimized method to extract poplar leaf proteins for two‐dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High‐quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE‐42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.