Protein extraction for two-dimensional electrophoresis from olive leaf,a plant tissue containing high levels of interfering compounds

The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.     

Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS

The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H₂O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2 % of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6 % of the metabolome by a combination of BD polar (59.4 %) and BD nonpolar (53.9 %) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).     

Development of an ultra-high performance liquid chromatography analytical methodology for the profiling of olive (Olea europaea L.) pulp proteins

Ultra-high performance liquid chromatography (UHPLC) constitutes an interesting proposal to speed protein separations but it is almost not explored. In this work UHPLC is proposed, for the first time, to separate olive pulp proteins. An important difficulty in the analysis of proteins is related to their extraction. The difficulty in the extraction of proteins from the olive pulp is derived from its high content in lipids and phenolic compounds. Eight different methods for the extraction of pulp proteins were designed and evaluated. The optimized extraction procedure consisted of a cleaning step to remove interfering compounds, followed by the extraction of proteins with a Tris–HCl buffer containing sodium dodecyl sulphate (SDS) and dithiothreitol (DTT), precipitation of proteins with acetone, and solubilization in the Tris–HCl buffer. This methodology yielded the most successful isolation of pulp proteins and enabled the optimization of a UHPLC methodology for their separation. The method was applied to the profiling of olive pulp proteins from different olive cultivars observing in all cases a protein that had never been described before.     

Influence of nonrubber components on properties of unvulcanized natural rubber

Low‐protein natural rubber (LPNR) and acetone‐extracted natural rubber (AENR) were prepared in solid form by alkaline treatment and acetone extraction to remove proteins and lipids. The content of proteins and lipids along with gel content were characterized by Fourier‐transform infrared spectroscopy (FTIR) and size exclusion chromatography with multiangle light scattering (SEC‐MALS) analysis. It was found that natural rubber (NR) treatment by alkaline hydrolysis or acetone extraction decreased proteins or lipids along with gel content. Also, having less proteins and lipids changed the network structure from macroaggregates to microaggregates. This resulted in inferior plasticity and poor mechanical, rheological, and dynamic properties. Furthermore, decreased strain‐induced crystallization and storage hardening were confirmed by temperature scanning stress relaxation (TSSR), after removal of proteins and lipids. Therefore, protein and lipid contents together with gel content play essential roles in controlling various properties of unvulcanized NR.     

A novel method of protein extraction from perennial Bupleurum root for 2-DE

The perennial Bupleurum root is thick and woody and contains high levels of interfering compounds. Common protein extraction methods have proved refractory towards the isolation of proteins suitable for 2-DE, due to the presence of interfering compounds. A novel method for extracting proteins suitable for 2-DE was established to overcome these problems. The main characteristic of this protocol is the partitioning of the proteins into the aqueous (fraction A-2), chloroform and isoamyl alcohol phases (A-3), and the interphase (A-1). The proteins are then extracted from each of these phases. From A-1, 85% (extracted protein against total proteins) proteins could be extracted and purified. For fraction A-2, a novel phenol extraction step is employed for the extraction of proteins. Based on the well-resolved 2-DE patterns, our protein preparation is free of interfering compounds. Using these methods (A-1, A-2, and A-3-3), a total of 3662 (1526 + 1128 + 1008) spots could be separated, and a protein yield of about 1.41 mg per 1.0 g fresh root material was obtained. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported that gives reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues as well.     

Modeling dimerizations of transmembrane proteins using Brownian dynamics simulations

The dimerizations of membrane proteins, Outer Membrane Phospholipase A (OMPLA) and glycophorin A (GPA), have been simulated by an adapted Brownian Dynamics program. To mimic the membrane protein environment, we introduced a hybrid electrostatic potential map of membrane and water for electrostatic interaction calculations. We added a van der Waals potential term to the force field of the current version of the BD program to simulate the short-range interactions of the two monomers. We reduced the BD sampling space from three dimensions to two dimensions to improve the efficiency of BD simulations for membrane proteins. The OMPLA and GPA dimers predicted by our 2D-BD simulation and structural refinement is in good agreement with the experimental structures. The adapted 2D-BD method could be used for prediction of dimerization of other membrane proteins, such as G protein-coupled receptors, to help better understanding of the structures and functions of membrane proteins.     

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.     

On-line SFE-GC for determination of PCBs in human milk and blood serum

On-line coupled supercritical fluid extraction and gas chromatography (SFE-GC) has been utilized for the determination of PCBs and other organochlorine compounds in human milk and blood serum. The procedure involved preconcentration of the sample on C₁₈-silica sorbent in an extraction cell: after precipitation of the proteins up to 20 ml of human milk was concentrated on 0.5 g of sorbent. Serum (up to 5 ml) was applied to the C₁₈ material without pretreatment. Basic alumina was utilized as a selective adsorbent for lipids in the on-line SFE-GC system. The method was used to analyze milk and serum spiked with 0.5 and 10 ng of Aroclor 1260 and the results compared with those obtained by liquid–liquid extraction of serum.     

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.     

Ionic Liquid-Based Extraction of Fatty Acids from Blue-Green Algal Cells Enhanced by Direct Transesterification and Determination Using GC × GC-TOFMS

Blue-green algae commonly referred to as cyanobacteria are known to grow in freshwater bodies when they are provided with suitable growth conditions such as nutrients, temperature and light. Algae biomass is known to contain a large amount of lipids, such as saturated and unsaturated fatty acids. In this study, fatty acids from algal cells were extracted using a newly developed extraction protocol using ionic liquid enhanced by direct transesterification at an elevated temperature. The identification and quantification of fatty acids was performed using gas chromatography coupled to a time-of-flight mass spectrometer (GC × GC-TOFMS). The extracted fatty acids were dominated by those with carbon chain of C16 and C18; [i.e. 7-hexadecenoic acid (C16:1) and hexadecanoic acid (C16:0) for C16, whereas C18 includes γ-linolenic acid (γ-C18:3); linoleic acid (C18:2); linolenic acid (C18:3); 6,9,12,15-octadecatetraenoic acid (C18:4); oleic acid (C18:1) and octadecanoic acid (C18:0)]. The obtained fatty acid composition was then compared with that obtained by organic solvent extraction using a mixture of chloroform and methanol. Statistical evaluation was performed using one-way ANOVA and found that there was no statistically significant difference (P = 0.908) between the two extraction methods, a finding which indicates the usefulness of ionic liquid as a solvent to replace volatile organic solvent to minimize environmental pollution.     

Extracellular Polymeric Substances (EPS) from Aerobic Granular Sludges: Extraction,Fractionation, and Anionic Properties

A multi-method protocol previously proposed for the extraction of extracellular polymeric substances (EPS) from flocculated sludges was investigated on dense aerobic granules. The protocol combines mechanical disruption by sonication and chemical extraction using the Tween detergent and the cation chelator, EDTA. Polysaccharides were mainly recovered during the first sonication step while proteins were recovered all along the extractive procedure with a high prevalence in the EDTA step. These data confirmed the interest of the multi-method protocol for harvesting a diversified pool of EPS from dense granules and for fractionation of the polymers according to their physicochemical properties. In addition, the high extractability of proteins with EDTA confers a specific behavior of the aerobic granules towards the multi-method extraction protocol, supporting the idea that proteins are associated in the granule matrix through ionic interactions involving divalent cations. Analysis of the extracted EPS by anionic exchange chromatography confirmed the presence of highly anionic proteins that were specifically detected in the extracts obtained from granules. One important question is now to investigate whether these highly anionic proteins are involved in the aggregation and densification process and if their presence is related to the cohesive properties of these particles.     

超高效液相色谱-三重四极杆质谱法同时测定美术颜料中33种游离态初级芳香胺

建立了超高效液相色谱-三重四极杆质谱法(UPLC-MS/MS)同时测定水粉画颜料、油画颜料、丙烯画颜料等美术颜料中33种初级芳香胺(PAAs)的检测方法。样品中的初级芳香胺用乙腈提取,经离心分离、氮吹浓缩后,以甲醇-水(1:9, v/v)定容至2 mL, 0.22 μm膜过滤后上机检测。采用BEH Phenyl柱(100 mm×2.1 mm, 1.7 μm),以含0.07%(v/v)甲酸的甲醇溶液-水(1:9, v/v)为流动相,梯度洗脱分离,UPLC-MS/MS多反应监测模式(MRM)检测,同位素内标法定量。方法优化了色谱分离条件、质谱碎裂电压、碰撞能量等,并考察了提取时间、提取溶剂、浓缩方式等对回收率的影响。33种初级芳香胺的方法检出限为5~50 μg/kg,定量限为15~150 μg/kg, 3种不同基质样品在3个添加水平的平均回收率为70.1%~115.8%,相对标准偏差(RSD)为2.1%~15%。本方法操作简便、快速、准确、灵敏度高,能满足相关测定的要求。     

Comparative evaluation of extraction methods for simultaneous mass-spectrometric analysis of complex lipids and primary metabolites from human blood plasma

Metabolomic results on human blood plasma largely depend on the sample preparation protocols employed for protein precipitation and metabolite extraction. Five different extraction methods were examined, which can be grouped into two categories, liquid-liquid extraction and protein precipitation methods, including long-standing protocols such as the Folch extraction and Bligh-Dyer extraction in comparison to modern methods such as the Matyash protocol and two global metabolite extraction methods. Extracts were subjected to analysis of blood plasma lipids and primary metabolites by using chip-based direct infusion nanoelectrospray tandem mass spectrometry and gas chromatography coupled to time-of-flight mass spectrometry, respectively. Optimal extraction schemes were evaluated based on the number of identified metabolites, extraction efficiency, compound diversity, reproducibility, and convenience for high-throughput sample preparations. Results showed that Folch and Matyash methods were equally valid and robust for lipidomic assessments while primary metabolites were better assessed by the protein precipitation methods with organic solvent mixtures. Graphical Abstract

Schematic workflow of five extraction methods and subsequent mass spectrometry analysis using GC-TOF MS and nanoelectrospray direct-infusion ion trap MS/MS?     

Cofactors in Biota: results of a German interlaboratory exercise on the determination of total lipids in fish tissue

 The content of total lipids is a common cofactor necessary for a normalisation of organic contaminant concentrations in biological materials. Lipids are routinely determined gravimetrically after extraction of the material with chlorinated solvents. A new method substituting chlorinated solvents for a cyclohexane/isopropanol mixture was to be evaluated by an intercomparison exercise. The participating laboratories were requested to determine total lipid content in three different samples of fish tissue following their own procedures as well as the new method described in a standard protocol. No significant differences in the overall means of total lipid content were found for the investigated samples regardless of the determination method. Using the harmonised protocol of the new method, between laboratory reproducibilities below 10% were obtained in the exercise. The results indicate the applicability of the harmonised method for the determination of total lipids as a cofactor in the analyses of contaminant concentrations in marine biota samples. Received: 27 June 2000 / Accepted: 20 September 2000     

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.     

The efficiency of alkaline extraction for the recovery of hexavalent chromium (CrVI) from paint samples and the effect of sample storage on CrVI recovery

Workplace exposures to CrVI, a human carcinogen, are significant in spraying operations of chromate-containing paints. Accurate determination of CrVI in paint aerosol air samples is important in assessing a worker's exposure to CrVI. National Institute for Occupational Safety and Health method 7604 is widely used for determining CrVI in air samples. It utilizes an alkaline extraction procedure. It was historically validated for paint aerosol samples containing 24.5 to 61.5 microg of CrVI. The literature documented potential airborne CrVI exposures greater than 61.5 microg in recent paint spraying operations. The efficiency of the alkaline method at extracting CrVI from paint samples containing 250 to 3000 microg of CrVI was determined. Paint was prepared, sampled, extracted twice and then digested. Extracts were analyzed for CrVI and digestates of the residual Cr were analyzed for total Cr. Alkaline extraction of paint samples using NIOSH method 7604 resulted in quantitative recoveries for paint samples with CrVI filter loadings from 250 to 3000 microg. A decrease in CrVI extraction efficiency was observed in samples containing > 1000 microg of CrVI. A second extraction improved the recovery of CrVI in these samples. Refrigerating paint aerosol samples for up to 2 weeks did not affect their CrVI content.     

Improved selection of LMW over HMW proteins from human plasma by mesoporous silica particles with external modification

Selective extraction of low molecular weight (LMW) proteins and peptides from complex biological samples plays an important role in the discovery of useful biomarkers and signaling molecules. Various methods, such as solid-phase extraction (SPE), ultrafiltration, and size-exclusion chromatography have been developed for such extraction purpose. In this study, we present, to our knowledge, the first demonstration of alkyl-diol@SiO₂ mesoporous material MCM-41 (alkyl-diol group on the external surface of mesoporous material) for selective extraction of LMW proteins and peptides from complex biological samples. The adsorption kinetics of LMW proteins, the influence of pH on adsorption and the desorption recovery by different elution solvents were investigated by using standard proteins as model samples. It was demonstrated that the modification of alkyl-diol group on the external surface could efficiently decrease the adsorption of HMW protein and increase the desorption recovery of LMW protein. Furthermore, the mesoporous materials were applied to selectively extract LMW proteins and peptides (<10 kDa) from crude human plasma. And the modified MCM-41 material had much better extraction selectivity and efficiency for LMW proteins and peptides than unmodified one.     

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.     

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.