Method development of efficient protein extraction in bone tissue for proteome analysis

Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases.     

Single unit filter-aided method for fast proteomic analysis of tear fluid

Human tear fluid is a complex mixture containing high concentrations of proteins and is increasingly becoming an important source for studying protein biomarkers of eye-related diseases such as Graves’ ophthalmopathy. Today, the Schirmer tear test is the most widely used technique for tear collection. However, sample handling and protein extraction from these strips have been highly challenging. Cutting and removal of the Schirmer strips after extraction, which may lead to sample loss prior to downstream analysis, are some of the challenges to consider. To address some of these limitations, we have developed a single-unit filter-aided method for both sample handling and protein extraction. In addition, we systematically investigated the most suitable conditions for protein extraction from these strips. Among the different extraction conditions applied, extraction with 100 mM ammonium bicarbonate containing 50 mM NaCl resulted in the highest number of identified proteins using one-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS). Moreover, 1526 proteins were identified when the optimized extraction method was combined with two-dimensional LC–MS/MS analysis, demonstrating the applicability of this novel approach to the study of the tear proteome. This dataset of identified proteins represents a comprehensive catalogue of the tear proteome and may serve as a list for future biomarker research.     

Methods for microbial DNA extraction from soil for PCR amplification

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.     

Extraction of wheat endosperm proteins for proteome analysis

Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.     

In Silico Characterization of Proteins: UniProt,InterPro and Integr8

Nucleic acid sequences from genome sequencing projects are submitted as raw data, from which biologists attempt to elucidate the function of the predicted gene products. The protein sequences are stored in public databases, such as the UniProt Knowledgebase (UniProtKB), where curators try to add predicted and experimental functional information. Protein function prediction can be done using sequence similarity searches, but an alternative approach is to use protein signatures, which classify proteins into families and domains. The major protein signature databases are available through the integrated InterPro database, which provides a classification of UniProtKB sequences. As well as characterization of proteins through protein families, many researchers are interested in analyzing the complete set of proteins from a genome (i.e. the proteome), and there are databases and resources that provide non-redundant proteome sets and analyses of proteins from organisms with completely sequenced genomes. This article reviews the tools and resources available on the web for single and large-scale protein characterization and whole proteome analysis.     

Comparison of extraction methods for the comprehensive analysis of mouse brain proteome using shotgun-based mass spectrometry

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-β-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.     

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.     

双向凝胶电泳比较三种常用蛋白质提取方法

组织(或细胞)的蛋白质提取效率直接影响蛋白质双向凝胶电泳(2-DE)的分辨率.为探索建立适用于人乳腺癌细胞株MCF-7蛋白质提取的最佳条件,比较目前在双向凝胶电泳中常用的3种蛋白质提取方法对MCF-7细胞总蛋白的提取效率.MCF-7细胞经培养后,分别采用M-PER试剂、标准裂解液或含硫脲裂解液提取其总蛋白质,然后进行双向凝胶电泳,并根据凝胶上蛋白质斑点的丰度和分布特点判断所得双向电泳图谱的质量,以确定MCF-7细胞蛋白质提取的相对最佳方法.结果显示,M-PER试剂法得到的图谱分辨率较低,蛋白质主要集中分布在分子量15~70kD,pH4.7~6.3的范围内;标准裂解液法得到的图谱分辨率有所提高,蛋白质分布比M-PER试剂法得到的图谱广;硫脲裂解液法得到的图谱是三者中分辨率最高的,尤其是高丰度蛋白和高分子量蛋白分离效果比前两者好.结果表明,在3种常用的蛋白质提取方法中,硫脲裂解液对细胞蛋白质的溶解性最佳,相对更适合于提取MCF-7细胞的蛋白质,并与双向凝胶电泳条件更兼容.     

Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.     

Microbial Protein in Soil: Influence of Extraction Method and C Amendment on Extraction and Recovery

The capacity to study the content and resolve the dynamics of the proteome of diverse microbial communities would help to revolutionize the way microbiologists study the function and activity of microorganisms in soil. To better understand the limitations of a proteomic approach to studying soil microbial communities, we characterized extractable soil microbial proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two methods were utilized to extract proteins from microorganisms residing in a Quitman and Benfield soil: (1) direct extraction of bulk protein from soil and (2) separation of the microorganisms from soil using density gradient centrifugation and subsequent extraction (DGC–EXT) of microbial protein. In addition, glucose and toluene amendments to soil were used to stimulate the growth of a subset of the microbial community. A bacterial culture and bovine serum albumin (BSA) were added to the soil to qualitatively assess their recovery following extraction. Direct extraction and resolution of microbial proteins using SDS-PAGE generally resulted in smeared and unresolved banding patterns on gels. DGC–EXT of microbial protein from soil followed by separation using SDS-PAGE, however, did resolve six to 10 bands in the Benfield but not the Quitman soil. DGC–EXT of microbial protein, but not direct extraction following the addition of glucose and toluene, markedly increased the number of bands (~40) on the gels in both Benfield and Quitman soils. Low recoveries of added culture and BSA proteins using the direct extraction method suggest that proteins either bind to soil organic matter and mineral particles or that partial degradation takes place during extraction. Interestingly, DGC may have been preferentially selected for actively growing cells, as gauged by the 10–100× lower cy19:0/18:1ω7 ratio of the fatty acid methyl esters in the isolated community compared to that for the whole soil. DGC can be used to isolate soil communities and provide microbial protein that can be characterized using PAGE.     

Quantitative proteomics to study the response of wheat to contrasting fertilisation regimes

Negative environmental impacts from mineral fertilisers and pesticides used in conventional cropping have raised concern over the sustainability of arable crop production. Organic cropping uses alternatives that avoid many of these negative environmental effects; however, crop yields can be significantly reduced, possibly due to a lower proportion of plant-available nutrients. To gain insights into the molecular effects of organic compared to conventional cropping systems on plant utilisation of nutrients, we used proteomics to analyse winter wheat (Triticum aestivum). Our aim was to investigate the effects of contrasting fertility management and crop protection regimes in organic and conventional cropping systems on the wheat flag leaf proteome and the association between the proteome and physiological traits. Wheat flag leaves were flash-frozen, lyophilised and milled prior to protein extraction (TCA/acetone) and analysed using 2D gel electrophoresis and MALDI-TOF MS. The abundance of 111 protein spots varied significantly between fertilisation regimes. Flag leaf N and P composition were significant drivers of differences in protein spot abundance, including major proteins involved in nitrogen remobilisation, photosynthesis, metabolism and stress response. These results indicate that molecular-based mechanisms are involved in the effect of contrasting cropping systems on nutrient utilisation and wheat grain yield. Using a functional genomics approach, we were able to identify proteins that are linked to causal genes, enabling the potential development of functional molecular markers for crop improvement in nutrient use efficiency.     

Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils

DNA extraction from soil samples is a critical step for molecular biology analyses. The present study compared the efficiency of two DNA isolation methods from non-polluted and polluted soils with or without the presence of a plant. Both applied methods used chemical and physical lyses, but method 1 had an additional physical disruption. The main difference between these two methods was the humic acid purification technique as it was carried out during cell lysis for method 1 and after cell lysis for method 2. Samples were assessed on the basis of their yield and DNA purity as well as their bacterial quantity and diversity. Based on our results, method 1 proved to be more effective at removing protein and RNA, whereas method 2 proved to be more effective at removing humic acids. Although no differences were obtained in terms of the DNA yield, both the bacterial quantity and community structure were affected by the method used. Method 1 allowed for the recovery of more information than method 2, and polluted soil was more sensitive to the DNA extraction procedure. We recommend carefully selecting the DNA extraction method, especially when soil is disturbed.     

Metaproteomics: Evaluation of protein extraction from activated sludge

Metaproteomic studies of full‐scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2‐step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B‐Per and bead beating. The data have been deposited to the ProteomeXchange with identifier PXD000862 ( http://proteomecentral.proteomexchange.org/dataset/PXD000862 ).     

Vaccinia virus-mediated high level expression and single step purification of recombinant Jak2 protein

Jak2 functions as a non-receptor tyrosine kinase and has been linked to pathologies such as cancer and cardiovascular disease. Because of this, many studies have tried to better understand its function. Unfortunately, very little information is known about its catalytic or biochemical properties as purification of significant amounts of functional Jak2 protein has been exceedingly difficult. Here, Jak2 was expressed in BSC-40 cells using a vaccinia virus-mediated expression system. Significant amounts ( approximately 10microg) of Jak2 protein were expressed from a single 100-mm cell culture dish. The protein was first harvested using three different methods of extraction to determine the relative efficiency of each lysis method with respect to Jak2 protein yield and catalytic activity. We found that lysis methods utilizing detergents increased the efficiency of protein extraction about 3-fold when compared to a method lacking detergent. However, with respect to catalytic activity, Jak2 isolated from cells using detergent-containing lysis buffers had significantly less catalytic activity than when compared to the method that was detergent free. Expression was then scaled up and Jak2 protein was purified via a one step immunoaffinity purification scheme using both the detergent-free and a modified detergent-containing method of extraction that maintained catalytic activity. In vitro kinase assays demonstrated that the purified product was highly catalytic as measured by its ability to tyrosine phosphorylate Stat1. Collectively, the results show that (1) Jak2 can be expressed at very high levels in mammalian cells, (2) it can be purified to homogeneity via a single step purification scheme, and (3) the purified product is biologically active.     

Lemon peel and Limoncello liqueur: A proteomic duet

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called “Limoncello”, stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.     

Genome-wide proteomics of Natronomonas pharaonis

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. According to genome analysis, the theoretical proteome consists of 2843 proteins. To reach further conclusions about its cellular physiology, the cytosolic protein inventory of Nmn. pharaonis has been analyzed using MS/MS on an ESI-Q-TOF mass spectrometer coupled on-line with a nanoLC system. The efficiency of this shotgun approach is illustrated by the identification of 929 proteins of which 886 are soluble proteins representing 41% of the cytosolic proteome. Cell lysis under denaturing conditions in water with subsequent separation by SDS-PAGE prior to nanoLC-MS/MS resulted in identification of 700 proteins. The same number (but a different subset) of proteins was identified upon cell lysis under native conditions followed by size fractionation (retaining protein complexes) prior to SDS-PAGE. Additional size fractionation reduced sample complexity and increased identification reliability. The set of identified proteins covers about 60% of the cytosolic proteins involved in metabolism and genetic information processing. Many of the identified proteins illustrate the high genetic variability among the halophilic archaea.     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。