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.     

Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.     

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.     

Optimizing protein recovery yield from serum samples treated with beads technology

Proteomics studies are often complicated by the wide dynamic range of the biological fluids, in which few highly abundant proteins obscure the signal of low abundant ones. To overcome this problem, several techniques have been developed on the basis of "depletion principles," namely immuno-subtraction with specific antibodies against the most-abundant proteins. Unfortunately, the probability of codepletion is a noteworthy drawback associated with these strategies. The ProteoMiner (PM) technology is a novel approach, consisting of a combinatorial library of hexapeptide ligands coupled to beads, that allows the capture of all species present in a proteome, but at much reduced protein concentration differences, simultaneously enhancing the concentration of the most dilute species. In this study, we evaluated the compatibility of the PM kit's elution reagent with 2-DE analysis, comparing five different purification methods on serum samples eluted from the beads: the "ReadyPrep 2-D Clean-up kit" and precipitation with organic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol. Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitation with ACN offered the most promising approach, showing the best spot resolution in all regions of the pH gradient and the greatest number of protein spots visualized on 2-D gels.     

Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.     

Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres

A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2 M NaCl, 100 mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100 ng mL⁻¹) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.     

Evaluation of the arsenic binding capacity of plant proteins under conditions of protein extraction for gel electrophoretic analysis

As prerequisite for the investigation of arsenic-binding proteins in plants, the general influence of different extraction parameters on the binding behaviour of arsenic to the plant protein pool was investigated. The concentration of the extraction buffer affected the extraction yield both for proteins and for arsenic revealing an optimal buffer concentration of 5 mM Tris/HCl, pH 8. The addition of 1 or 2% (w/v) SDS to the extraction buffer produced a two- to threefold enhancement of the total protein extraction yield but strongly suppressed the simultaneous extraction of arsenic from 80 ± 8% extraction yield obtained without SDS to 48 ± 2% in presence of 2% (w/v) SDS. The arsenic binding capacity of the protein fraction obtained after extraction with Tris buffer and protein precipitation by trichloroacetic acid in acetone was estimated to be 1.4 ± 0.6% independently on the original spiking concentration of arsenic provided in the form of monomethylarsonate to the extracts. Due to the low total protein concentrations of the plant extracts that varied in the range from 75 to 412 μg mL⁻¹ depending on the extraction parameters, high arsenic concentrations of 263-1001 mg (kg protein mass)⁻¹ resulted for spiking concentrations of 10 mg As L⁻¹. The optimized protein isolation procedure was applied to plants grown under arsenic exposure and revealed a similar arsenic binding capacity as for the spiked protein extracts.     

Effect of physicochemical conditions on the ultrafiltration of β-lactoglobulin: Fluorescence probing of induced structural changes

This work aims for determining the impact of different environmental conditions, such as pH, ionic strength (salt concentration) and the chemistry of the membrane surface (hydrophilic/hydrophobic character) on the structure of permeating proteins after ultrafiltration. In the permeation experiments reported in this paper, different solutions of a model protein – β-lactoglobulin – at pH 3, 5 and 8 and salt concentrations of 1, 10 and 100 mM were processed with membranes of different molecular weight cut-off (10 and 30 kDa) and materials (regenerated cellulose—RC, and polyethersulfone—PES).     

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.     

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.     

Plucking,pillaging and plundering proteomes with combinatorial peptide ligand libraries

Recent developments in the technique of combinatorial peptide ligand libraries, for enhancing the visibility of the low-abundance proteome, are reviewed here. Novel en bloc elution systems, allowing essentially complete proteome recovery in a single step, are reported here, particularly, en bloc elution with 3–5% boiling sodium dodecyl sulphate (SDS) or in urea–thiourea–CHAPS added with either 40 mM formic acid or 25 mM cysteic acid. Novel capturing systems are also discussed: in particular, although capturing at pH 7.2 in physiological saline has always been recommended, it is shown that capturing also at acidic (pH 3.8) and alkaline (pH 9.5) values substantially increments the total captured protein population. Some examples of detection of novel proteins by the described methodology are also discussed. In particular, in the case of venom proteins, where essentially all components had been detected and fully described by conventional means, the application of the ligand library technology allowed the discovery of two, previously unreported, trace enzymes necessary for the maintenance of the native structure of venom components, namely peroxiredoxin and glutaminyl cyclase. In the case of the red blood cell (RBC) cytoplasmic proteome, where a grand total of 1570 components of the 2% minority proteomes have been identified, these findings allowed to unravel the genetic defect of a rare RBC disease, called congenital dyserythropoietic anemia type II. The mutations are located in the SEC23B gene coding for the SEC23B protein, detected for the first time in the RBC proteome thanks to the peptide capturing technology.     

A green deep eutectic solvent-based aqueous two-phase system for protein extracting

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.     

imFASP: An integrated approach combining in-situ filter-aided sample pretreatment with microwave-assisted protein digestion for fast and efficient proteome sample preparation

An integrated sample preparation method, termed “imFASP”, which combined in-situ filter-aided sample pretreatment and microwave-assisted trypsin digestion, was developed for preparation of microgram and even nanogram amounts of complex protein samples with high efficiency in 1 h. For imFASP method, proteins dissolved in 8 M urea were loaded onto a filter device with molecular weight cut off (MWCO) as 10 kDa, followed by in-situ protein preconcentration, denaturation, reduction, alkylation, and microwave-assisted tryptic digestion. Compared with traditional in-solution sample preparation method, imFASP method generated more protein and peptide identifications (IDs) from preparation of 45 μg Escherichia coli protein sample due to the higher efficiency, and the sample preparation throughput was significantly improved by 14 times (1 h vs. 15 h). More importantly, when the starting amounts of E. coli cell lysate decreased to nanogram level (50–500 ng), the protein and peptide identified by imFASP method were improved at least 30% and 44%, compared with traditional in-solution preparation method, suggesting dramatically higher peptide recovery of imFASP method for trace amounts of complex proteome samples. All these results demonstrate that the imFASP method developed here is of high potential for high efficient and high throughput preparation of trace amounts of complex proteome samples.     

Effect of protein aggregates on foaming properties of β-lactoglobulin

Our paper aims at determining the respective part of protein aggregates and non-aggregated proteins in the foam formation and stability of β-lactoglobulin. We report results on fractal aggregates formed at neutral pH and strong ionic strength (aggregates size from 30 to 190 nm). Pure aggregates and mixtures of non-aggregated/aggregated proteins at varying ratios were used. The capacity of aggregates to form and stabilize foams has been studied in relation with their ability to absorb at air/water interfaces. Our results show that protein aggregates are not able by themselves to improve the foaming properties but participate to a better foam stabilization in the presence of non-aggregated proteins. Non-aggregated proteins appear to be necessary to produce stable foams. We have shown that the amount and the size of aggregates had an influence on the drainage rate.     

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by ¹H nuclear magnetic resonance (¹H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV–vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV–vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins.     

Extraction mechanism of sulfamethoxazole in water samples using aqueous two-phase systems of poly(propylene glycol) and salt

Based on the poly(propylene glycol)₄₀₀ (PPG₄₀₀)–salt aqueous two-phase system (ATPS), a green, economical and effective sample pretreatment technique coupled with high performance liquid chromatography was proposed for the separation and determination of sulfamethoxazole (SMX). The extraction yield of SMX in PPG₄₀₀–salt ATPS is influenced by various factors, including the salt species, the amount of salt, pH, and the temperature. Under the optimum conditions, most of SMX was partitioning into the polymer-rich phase with the average extraction efficiency of 99.2%, which may be attributed to the hydrophobic interaction and salting-out effect. This extraction technique has been successfully applied to the analysis of SMX in real water samples with the recoveries of 96.0–100.6%, the detection limits of 0.1 μg L⁻¹, and the linear ranges of 2.5–250.0 μg L⁻¹.     

Method development for proteome stabilization in human saliva

Human saliva is a biological fluid with emerging early detection and diagnostic potentials. However, the salivary proteome suffers from rapid degradation and thus compromises its translational and clinical utilities. Therefore, easy, reliable and practical methods are urgently required for the storage of human saliva samples. In this study, saliva samples from healthy subjects were collected and stored at room temperature (RT) and 4 °C for different lengths of time with and without specific protein stabilization treatments. SDS-PAGE was run to compare the protein profiling between samples. Reference proteins, β-actin and interleukin-1 β (IL1β), were chosen to evaluate salivary protein stability. Immunoassay was used for the detection of these target proteins. All data was compared with the positive control that had been kept at −80 °C. The results show that the salivary proteome that has been stored at 4 °C with added protease inhibitors was stable for approximately two weeks without significant degradation. By adding ethanol to the samples, the salivary proteome was stabilized at RT. After optimization, a simple, robust and convenient method is developed for the stabilization of proteins in human saliva that does not affect the downstream translational and clinical applications. The salivary proteome could be stabilized without significant degradation by adding ethanol at RT for about two weeks. This optimized method could greatly accelerate the clinical usage of saliva for future diagnosis.     

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns.     

Increasing proteome coverage for gel‐based human tear proteome maps: towards a more comprehensive profiling

In situations where the molecular mechanism of many ocular disorders is unknown, owing to the difficulties associated with sampling from ocular tissues, human tear film can be a promising medium in ophthalmic research. The present study demonstrates an in‐depth gel‐based proteome optimization survey to approach more appropriate and efficient systems in various situations such as normal and dry‐eye syndromes. Therefore, systematic and statistical evaluations were performed on different preparation methods, including acetone, acetone–methanol, chloroform–methanol–water, trichloroacetic acid (TCA)–acetone, tri‐n‐butylphosphate–acetone–methanol precipitations and ammonium sulfate fractionation at three different percentages of saturations (50, 70 and 90%). Methods were compared quantitatively on both one‐ and two‐dimensional patterns. Some important parameters such as total protein recovery yield, densitometric analysis of some protein contaminants, banding patterns and total spot numbers along with statistical models for proper clustering were considered. Findings revealed noticeable impacts of preparation methods on all aspects of gel‐based separations as well as recovery yield (ranging from 5.29 ± 0.96 to 22.56 ± 1.77 µg/mm) and banding and pattern resolution. In addition to all these, the most important point is that the total protein spot number on the final two‐dimensional patterns (varied from 528.00 ± 19.00 to 657.00 ± 21.52 for different methods) were also noticeably increased in comparison with previously published reports (maximum of 250 spots), which is essential for a more comprehensive analysis. Increasing the proteome coverage in the present study is supposed to originate from improved solubility and effective rehydration during the sample application and isoelectric focusing (IEF) procedure along with proper sample preparation. Copyright © 2014 John Wiley & Sons, Ltd.     

An automatic system for multidimensional integrated protein chromatography

An automatic system for multidimensional integrated protein chromatography was designed for simultaneous separation of multiple proteins from complex mixtures, such as human plasma and tissue lysates. This computer-controlled system integrates several chromatographic columns that work independently or cooperatively with one another to achieve efficient high throughputs. The pipelines can be automatically switched either to another column or to a collection container for each UV-detected elution fraction. Environmental contamination is avoided due to the closed fluid paths and elimination of manual column change. This novel system was successfully used for simultaneous preparation of five proteins from the precipitate of human plasma fraction IV (fraction IV). The system involved gel filtration, ion exchange, hydrophobic interaction, and heparin affinity chromatography. Human serum albumin (HSA), transferrin (Tf), antithrombin-III (AT-III), alpha 1-antitrypsin (α1-AT), and haptoglobin (Hp) were purified within 3 h. The following recovery and purity were achieved: 95% (RSD, 2.8%) and 95% for HSA, 80% (RSD, 2.0%) and 99% for Tf, 70% (RSD, 2.1%) and 99% for AT-III, 65% (RSD, 2.0%) and 94% for α1-AT, and 50% (RSD, 1.0%) and 90% for Hp. The results demonstrate that this novel multidimensional integrated chromatography system is capable of simultaneously separating multiple protein products from the same raw material with high yield and purity and it has the potential for a wide range of multi-step chromatography separation processes.