New method for prefractionation of plasma for proteomic analysis

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high‐abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high‐abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow? technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2‐DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.     

An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE

Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues.     

Alteration of protein profile in rat liver of animals exposed to subacute diazinon: A proteomic approach

Diazinon, an organophosphorus insecticide, is employed to control pests in agriculture. Diazinon may contaminate the environment during the manufacturing process or agricultural application. Previous studies have revealed that diazinon may induce alteration in the protein profile of the liver. Here, a proteomics approach was used to investigate the effects on the protein profile in the liver of rats of subacute oral exposures at 15 mg/kg of diazinon. Liver proteins were separated using 2D‐PAGE, and stained by MS‐compatible silver staining and/or the fluorescent SYPRO® Ruby protein gel stain. Gels were scanned and analyzed using the Image Master software. Differentially displayed protein species were identified using MALDI‐TOF/TOF and MASCOT software. Significantly altered protein species were identified to be involved in apoptosis, cell metabolism, transport, and antioxidant systems. Exposure to diazinon decreased levels of some species of catalase, peroxiredoxin‐6, 3‐ketoacyl‐CoA thiolase, and glucose regulated protein78, whereas the level of protein disulfide‐isomerase A3 increased. Our results suggested that diazinon may induce hepatotoxicity through oxidative stress, apoptosis, and metabolic disorders in rat liver.     

An improved pixel-based approach for analyzing images in two-dimensional gel electrophoresis

An improved pixel-based approach for analyzing 2-DE images is presented. The key feature of the method is to create a mask based on all gels in the experiment using image morphology, followed by multivariate analysis on the pixel level. The method reduces the impact of noise and background by identifying regions in the image where protein spots are present, but make no assumption on individual spot boundaries for isolated spots. This makes it possible to detect significant changes in complex regions, and visualize these changes over multiple gels in an easy way. False missing values and spot volumes caused by imposing erroneous spot boundaries are thus circumvented. The approach presented gives improved pixel-based information from the gels, and is also an alternative to existing methods for data-reduction, significance testing and visualization of 2-DE data. Results are compared with software using a common spot boundary approach on an experiment consisting of 35 full size gel images. Gel alignment is required before analysis.     

2-DE proteomic analysis of the model cyanobacterium Anabaena variabilis

Cyanobacteria are photosynthetic bacteria capable of producing hydrogen and secondary metabolites with potential pharmaceutical applications. A limited number of cyanobacterial 2-DE proteomic studies have been published, most of which are based on Synechocystis sp. PCC 6803. Here, we report the use of 2-DE, ESI-MS/MS and protein bioinformatics tools to characterize the proteome of Anabaena variabilis ATCC 29413, a heterocystous nitrogen-fixing cyanobacterium that is a model organism for the study of nitrogen fixation. Using a 2-DE workflow that included the use of a detergent-based extraction buffer and 3-10 nonlinear IPG strips resulted in the identification of 254 unique proteins, with significantly better coverage of basic and low-abundance proteins that has been reported in 2-DE analyses of Synechocystis sp. A set of protein bioinformatics tools was employed to provide estimates of protein localization, hydrophobicity, abundance and other properties. The characteristics of the A. variabilis proteins identified in this study were compared against the theoretical proteome for this organism, and more generally within the cyanobacteria, to identify opportunities for further development of 2-DE-based cyanobacterial proteomics.     

Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysis

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.     

Full-fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3-D analytical technique: Identification of new heterodimeric aggregation states

Wheat proteinaceous alpha-amylase inhibitors (alpha-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive alpha-AIs that share sequence homology, but differ in their specificity against alpha-amylases from different species and for their aggregation states. Wheat alpha-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive alpha-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of alpha-AIs (resistant to T > 70 degrees C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the alpha-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric alpha-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric alpha-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat alpha-AIs.     

Comparative analysis of proteomic changes in contrasting flax cultivars upon cadmium exposure

Cadmium (Cd) is classified as a serious pollutant due to its high toxicity, high carcinogenicity, and widespread presence in the environment. Phytoremediation represents an effective low‐cost approach for removing pollutants from contaminated soils, and a crop with significant phytoremediation potential is flax. However, significant differences in Cd accumulation and tolerance were previously found among commercial flax cultivars. Notably, cv. Jitka showed substantially higher tolerance to elevated Cd levels in soil and plant tissues than cv. Tábor. Here, significant changes in the expression of 14 proteins (related to disease/defense, metabolism, protein destination and storage, signal transduction, energy and cell structure) were detected by image and mass spectrometric analysis of two‐dimensionally separated proteins extracted from Cd‐treated cell suspension cultures derived from these contrasting cultivars. Further, two proteins, ferritin and glutamine synthetase (a key enzyme in glutathione biosynthesis), were only up‐regulated by Cd in cv. Jitka, indicating that Cd tolerance mechanisms in this cultivar may include maintenance of low Cd levels at sensitive sites by ferritin and low‐molecular weight thiol peptides binding Cd. The identified changes could facilitate marker‐assisted breeding for Cd tolerance and the development of transgenic flax lines with enhanced Cd tolerance and accumulation capacities for phytoremediating Cd‐contaminated soils.     

A protein extraction method compatible with proteomic analysis for the euhalophyte Salicornia europaea

Protein extraction from plants like the halophyte Salicornia europaea has been problematic using standard protocols due to high concentrations of salt ions in their cells. We have developed an improved method for protein extraction from S. europaea, which allowed us to remove interfering compounds and salt ions by including the chemicals borax, polyvinylpolypyrrolidone, and phenol. The comparative study of this method with several other protocols using NaCl-treated S. europaea shoots demonstrated that this method gave the best distinction of proteins on 2-DE gels. This protocol had a wide range of applications as high yields and good distinction of 1-DE gels for proteins isolated from twelve other plants were rendered. In addition, we reported results of 2-DE using the recalcitrant tissue of the S. europaea roots. We also demonstrated that this protocol is compatible with proteomic analysis as eight specific proteins generated by this method have been identified by MS. In conclusion, our newly developed protein extraction protocol is expected to have excellent applications in proteomic studies of halophytes.     

Systematic comparison of technical details in CBB methods and development of a sensitive GAP stain for comparative proteomic analysis

Considering the importance of CBB staining in visualizing proteins in 2-DE gels, any improvement in the existing protocols with high sensitivity and good MS compatibility is of significant importance. In this study, we systematically evaluated the effects of different staining parameters on CBB methods by 1-DE and 2-DE, and demonstrated that G-250 was more suitable for visualizing low-abundant proteins as well as generating more spots than R-250, whereas R-250 had a superior capability for quick staining of high-abundant proteins. The staining produced by mixing G-250 and R-250 in different ratios showed similar sensitivity. Compared with acetic acid, phosphoric acid produced more protein spots. Ammonium-based stain demonstrated a superior sensitivity than the aluminum-based one. Based on these findings, a new protocol using CBB G-250, ammonium sulfate and phosphoric acid (GAP) was developed by incorporating the fixation, sensitization and staining procedures together. The comparison of GAP with other methods revealed that GAP generated more protein spots and had wider applications. The identification of 11 proteins demonstrated that GAP was not only compatible with MS but also obviously reduced in vitro protein modification, and thus could be a preferable protocol in the future proteomic analysis.     

Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added it substitutes the analyte in the antibody complex, but not the blocking reagent. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 0.2 nmol l⁻¹) obtained by the above method was 10 times lower than that obtained by the corresponding ELISA. As non-competitive immunoassays reported for small molecules up to now have been no more than just approaches, the suitability of the proposed assay for cortisol quantification in a real matrix was investigated. Human saliva was chosen as a matrix because of the need for very sensitive techniques to determine salivary cortisol content. The matrix effect was offset by performing the calibration experiments in acidic conditions (pH=5.6) and adding 0.1% of bovine serum albumin (BSA) to the buffer. In these conditions, the LOD was 1.4 nmol l⁻¹, which was adequate to measure normal levels of cortisol. Spiked samples were analysed and gave recoveries ranging from about 80 to 120%. Therefore, five subject samples, collected over 18 h showed salivary cortisol concentrations compatible with the circadian variation of reported normal values.     

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 μL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9 h⁻¹, a linear calibration range of 0.5–15 mg L⁻¹ (absorbance = 5.40 × 10⁻² [acetaldehyde, mg L⁻¹], R² = 0.998), a relative standard deviation (RSD) of 1.90% (n = 10, acetaldehyde concentration of 2.5 mg L⁻¹), and a limit of detection (LOD) of 12.3 μg L⁻¹. The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD = 93.0 μg L⁻¹ and sampling rate = 4 h⁻¹). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6–101.9%), comparable to those obtained by GC (96.4–102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.     

Application of capillary zone electrophoresis to the analysis and to a stability study of nitrite and nitrate in saliva

The applicability of capillary zone electrophoresis for the determinations of nitrite and nitrate was studied. Using direct UV detection the limit of detection values of the analytes were 0.14 and 0.21 microg/mL, respectively. The developed method was found to be useful to directly determine nitrite, nitrate and thiocyanate in saliva. It was found that adjusting the pH of the sample to 11 and storing the saliva at 4 degrees C was adequate to make constant the nitrite/nitrate ratio in saliva samples at least 7 days.     

An inter-laboratory comparison for determination of cortisol in saliva

An inter-laboratory comparison study for cortisol in saliva is reported in the present study. Nine laboratories representing four different methods participated in the study. Each laboratory measured five blind samples prepared from natural saliva spiked with a pure certified reference material in the range 0–70 nmol/L. The average observed values were established as means of eight laboratories, because one laboratory was excluded as an outlier. The natural content of cortisol in the pooled saliva was 6.8 nmol/L. The recovery of the certified reference material was 83%–131% (164% for the outlying laboratory). For five laboratories the 95%-confidence intervals of the recoveries did not cover 100%. The standard deviations for a concentration of 10 nmol/L were estimated for each laboratory. They were 0.64–1.67 nmol/L (6.26 nmol/L for the outlying laboratory). The present study emphasizes the need for an external proficiency-testing scheme and a certified reference material for cortisol in saliva. Received: 8 May 2002 Accepted: 3 September 2002     

Trends in proteomic analysis of human vitreous humor samples

Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low‐abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel‐based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set‐ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

Two dimensional electrophoretic analysis of human tears: collection method in dry eye syndrome

Tear proteomics, by 2-DE, can give a fingerprint of the protein profile, which is well suited in clinical proteomics for biomarker identification and in diagnostics. The mode of tear collection can influence the representation of the proteins in the tear and therefore it is important to use the appropriate method. In this study, capillary and Schirmer mode of tear collection was done in the healthy controls and the Schirmer method was validated in dry eye syndrome conditions. 2-D PAGE of normal and dry eye tear was performed using pH 3-10 linear IPG strips followed by 13% SDS-PAGE. The spot intensity was analyzed by the PD quest software. The two methods were compared using Bland-Altman statistical tool. The 2-D map of capillary and Schirmer tear showed 147 ± 8 spots and 145 ± 7 spots respectively. Both the collection methods were in agreement with each other and were comparable. Dry eye tear protein showed differential expression of proteins as observed in 25-35 kDa region. One of the significantly reduced protein was identified as proline-rich 4 protein. Schirmer method of tear collection is reliable in patients with dry eye, which can display the differential protein expression and help in biomarker identification.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

Genetic analysis of human complement factor H polymorphisms

Human complement factor H (factor H) is polymorphic, with five previously reported FH alleles and three previously reported HF alleles (HF*A, HF*B, and HF*Q0). The relationship between the FH and HF alleles is not clear, and the genetic basis of factor H phenotypes has not yet been identified. In this study, nucleotide sequence analysis of complementary DNA (cDNA) from individuals with each HF phenotype identified seven mutated sites in the factor H gene. However, in four cases, the same cDNA sequence was observed in individuals with two different HF phenotypes. Western blotting and 2-DE also showed that a 160 kDa protein corresponding to factor H was expressed in individuals with HF phenotypes. In addition, factor H cross-reacting 45 and 42 kDa polypeptides were detected in individuals with HF A, HF B, or HF AB phenotypes, but not in individuals with the HF Q0 (a null allele) phenotype. Thus, HF phenotype did not correlate well with factor H gene or protein structural variation. Evidence is provided to support the hypothesis that the HF phenotypes do not correspond to polymorphism in factor H, but instead correspond to polymorphism in factor H-related protein 1. A novel PCR-RFLP method was developed and used to detect four polymorphisms (G257A, G1492A, A2089G, and G2881T) in the factor H gene in 54 unrelated Japanese individuals. This method could be useful for studies on genetic disease associated with these mutations.