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.     

Proteome profile of salt gland‐rich epidermis extracted from a salt‐tolerant tree species

Preparation of proteins from salt‐gland‐rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high‐quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland‐rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland‐rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol‐based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D‐LC‐MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol‐based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high‐throughput proteomic analyses involving LC‐MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.     

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。     

Evaluation of extraction procedures for 2‐DE analysis of aphid proteins

Protein sample preparation is a crucial step in a 2‐DE proteomics approach. In order to establish a routine protocol for the application of proteomics analysis to aphids, this study focuses on the specific protein extraction problems in insect tissues and evaluates four methods to bypass them. The approaches of phenol extraction methanol/ammonium acetate precipitation (PA), TCA/acetone precipitation, PEG precipitation, and no precipitation were evaluated for proteins isolation and purification from apterous adult aphids, Sitobion avenae. For 2‐DE, the PA protocol was optimal, resulting in good IEF and clear spots. PA method yielded the greatest amount of protein and displayed most protein spots in 2‐DE gels, as compared with the TCA/acetone precipitation, PEG precipitation and no precipitation protocols. Analysis of protein yield, image quality and spot numbers demonstrate that the TCA/acetone precipitation protocol is a reproducible and reliable method for extracting proteins from aphids. The PEG precipitation approach is a newly developed protein extraction protocol for aphids, from which more unique protein spots can be detected, especially for detection of acid proteins. These protocols are expected to be applicable to other insects or could be of interest to laboratories involved in insect proteomics, despite the amounts and types of interfering compounds vary considerably in different insects.     

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.     

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.     

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.     

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.     

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.     

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.     

Combination of virtual and experimental 2DE together with ESI LC‐MS/MS gives a clearer view about proteomes of human cells and plasma

Virtual and experimental 2DE coupled with ESI LC‐MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI‐TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC‐MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw ( http://web.expasy.org/compute_pi/pi_tool‐doc.html ). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.     

Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of Gas Chromatography‐Tandem Mass Spectrometry (GC‐MS‐MS) with Pulsed Splitless Injection for the Determination of Multiclass Pesticides in Vegetables

Abstract

GC‐MS‐MS was used to analyze vegetables for 14 pesticide residues. In the same run, the optimal ionization mode (electron ionization or chemical ionization) for each pesticide was selected. The method includes a simple extraction procedure with acetone and dichloromethane. A solid‐phase extraction tube was used for cleanup. The 1 µL of the final solution was injected into the instrument by pulsed splitless injection mode that can improve the sensitivity. The average recoveries were ranged from 78% to 102%. The relative standard deviation was lower than 18.4%. The calculated limits of detection were equal or lower than 0.02 mg/kg.     

Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis

Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein–DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA–protein complexes, due to strong Coulombic repulsion.     

Optimization and validation of organochlorine compounds in adipose tissue by SPE‐gas chromatography

Scientific evidence has shown an association between organochlorine compounds (OCC) exposure and human health hazards. Concerning this, OCC detection in human adipose samples has to be considered a public health priority. This study evaluated the efficacy of various solid‐phase extraction (SPE) and cleanup methods for OCC determination in human adipose tissue. Octadecylsilyl endcapped (C₁₈‐E), benzenesulfonic acid modified silica cation exchanger (SA), poly(styrene‐divinylbenzene (EN) and EN/RP₁₈ SPE sorbents were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture detection (GC–ECD). The C₁₈‐E columns with strong homogenization were found to provide the most effective cleanup, removing the greatest amount of interfering substance, and simultaneously ensuring good analyte recoveries higher than 70%. Recoveries > 70% with standard deviations (SD) < 15% were obtained for all compounds under the selected conditions. Method detection limits were in the 0.003–0.009 mg/kg range. The positive samples were confirmed by gas chromatography coupled with tandem mass spectrometry (GC‐MS/MS). The highest percentage found of the OCC in real samples corresponded to HCB, o,p′‐DDT and methoxychlor, which were detected in 80 and 95% of samples analyzed respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Multidimensional separation of tryptic peptides from human serum proteins using reversed‐phase,strong cation exchange,weak anion exchange,and fused‐core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high‐abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C₁₈ at pH 2.5 (low pH), fused‐core fluorinated at pH 2.5, and RP C₁₈ at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip‐LC–MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low‐pH RP chromatography. A 3D separation scheme combining ERLIC, high‐pH RP, and low‐pH nanoChip‐LC–MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.     

Comparison of protein precipitation methods from adipose tissue using difference gel electrophoresis

Proteomic methods have great potential to aid our understanding of the functional and pathological roles of adipose tissue. A critical initial step in the proteomic studies is the efficient isolation of proteins before conducting detailed analysis. In this study, three different methods were used for precipitating proteins; we analyzed samples from visceral adipose tissue, subcutaneous adipose tissue, and stromal visceral fraction extracts after chloroform/methanol, acetone, and trichloroacetic acid precipitation. The proteins recovered after the precipitation steps were examined by 2D‐DIGE. Statistical analyses were carried out using simple linear regression analyses and R2 values were calculated for the intra‐ and inter‐method comparisons. We found that all three precipitation methods provided highly reproducible protein spots that were recovered when run in duplicate using the same method of precipitation, irrespective of whether it was solvent (R2 = 0.85–0.98) or acid‐based (R2 = 0.80–0.96). A higher variation and poor correlation was noted for the recovered protein spots when comparisons were made between the methods (R2 = 0.40–0.88) and also when the same method was compared between different sample types. In this study, TCA‐precipitated samples were enriched in lower molecular mass proteins compared to the samples extracted by solvent‐based precipitation methods.     

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno‐capture of ProGRP in 96‐wells microtiter plates coated with the mAb E146. After immuno‐capture and thorough rinse, trypsin was added for in‐well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC‐MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9–17.5%), accuracy (3.0–6.6%), and linearity (r² = 0.995) in the tested range (0.5–50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC‐MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno‐capture LC‐MS method was superior with respect to sensitivity. This illustrates the large potential of immuno‐capture sample preparation prior to LC‐MS in protein biomarker quantification.     

Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips,stems, roots and leaves

The flavonoids and anthocyanins in Chinese sugarcane (Saccharum sinensis Roxb.) tips, stems, roots and leaves were separately analyzed by HPLC‐UV diode array detector, respectively. The results indicated that the content of flavonoids in sugarcane leaves was considerably high in comparison with previous reports in Brazil sugarcane (S. officinarum L.) leaves. Moreover, the content of flavonoids in sugarcane tips and roots was also high in comparison with sugarcane stems. For another, the content of anthocyanins in sugarcane roots was higher than that in other parts of the sugarcane, such as leaves, tips and stems. In addition, two anthocyanins, named petunidin 3‐O‐(6″‐succinyl)‐rhamnoside and cyanidin‐3‐O‐glucoside, were first identified from S. sinensis by HPLC‐UV diode array detector and HPLC‐MS/MS.     

Simultaneous determination of multiresidual phenyl acetanilide pesticides in different food commodities by solid‐phase cleanup and gas chromatography‐mass spectrometry

An efficient and sensitive multiresidue method has been developed for quantification and confirmation of 25 phenyl acetanilide pesticides in a wide variety of food commodities including maize, spinach, mushroom, apple, soybean, chestnut, tea, beef, cattle liver, chicken, fish, and milk. Analytes were extracted with acetone–n‐hexane (1:2, v/v) followed by cleanup using SPE. Several types of adsorbents were evaluated. Neutral aluminum and graphitized carbon black cartridge showed good cleanup efficiency. The extract was determined by GC‐MS in the selected ion monitoring mode using one target and two qualitative ions for each analyte. The limits of detection were 0.01 mg/kg for all analytes. The average recoveries ranged from 66.9 to 110.6% (mean 88.8%) and RSDs were in the range 2.0–19% (mean 10.5%) across three fortification levels. The proposed method was successfully applied to real samples in routine analysis and a satisfactory result was obtained.